Selective high-affinity polydentate ligands and methods of making such

ABSTRACT

This invention provides novel polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each bind different region son the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/988,801, filed Feb. 3, 2011, which is a national phase applicationunder 35 U.S.C. §371 of International Application No. PCT/US2009/041276,filed Apr. 21, 2009, which in turn claims priority under 35 U.S.C.§119(e) to U.S. Provisional Application No. 61/046,712, filed Apr. 21,2008, the content of each of which is incorporated herein by referencein its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This work was supported in part by grant No: CA047829 from the NationalInstitutes of Health, National Cancer Institute. In addition, pursuantto Contract No: DE-AC52-07NA27344 between the United States Departmentof Energy and Lawrence Livermore National Security LLC for the operationof Lawrence Livermore National Laboratory, the Government of the UnitedStates of America has certain rights in this invention.

FIELD

This invention pertains to the development of targeting molecules. Moreparticularly this invention pertains to the development of polydentateselective high affinity ligands (SHALs) that can be used in a manneranalogous to antibodies and/or peptide ligands as affinity reagents orfor the diagnosis and treatment of various diseases (e.g., cancer).

BACKGROUND

In general, it has been found that cancericidal drugs, such aschemotherapeutics, are also toxic to cells of normal tissues.Consequently, the side effects of such drugs can be almost asdevastating to the patient as the malignant disease itself. The adventof monoclonal antibodies and peptide ligands provided a new means forimproving drug specificity/selectivity. By conjugating, e.g., acytotoxic agent to an antibody or peptide ligand directed againstantigens present on malignant cells, but not present on normal cells,selective killing of malignant cells has been achieved. Many differentimmunoconjugates comprising an antibody attached to a cytotoxic agenthave been created directed against a variety of cell-surface antigens.

Cytotoxic agents used in such immunoconjugates include radioisotopes,various plant and bacterial toxins (e.g., Pseudomonas exotoxin,diphtheria toxin, ricin, abrin, etc.), various growth factors, and morerecently, agents such as caspases. Although there have been somesuccesses, notably in lymphoma and leukemias, antibody-based therapywhether using antibodies alone or immunoconjugates, has generally notfulfilled the expected potential.

Although significant advances have been made in the treatment ofmalignant disease, curative regimens for most patients have not yet beendeveloped or are associated with toxicities unattractive for thepatient. Therefore, new strategies for the treatment of most malignantdiseases are needed. These strategies should have as their goal, themaximization of therapeutic effect, coupled with the minimization oftoxicity. One approach has involved the use of ligands specific for cellsurface receptors or antibodies specific for malignant cell associatedantigens as a means of targeting drugs or radioisotopes to the malignantcells. The approach is attractive for many malignant diseases becausethe malignant cells display a variety of tumor-restricted or upregulatedantigens and/or receptors on their cell surfaces which would beavailable for targeting. Thus far, antibody/antigen systems have beenfound to be better than ligand receptor systems because they are morerestricted than receptors and in greater abundance on the malignantcell.

Despite these advantages, antibodies have not fulfilled their potentialfor many reasons. Among the reasons, antibodies are macromolecules(large molecules) that often do not effectively access and penetrate themalignant tumor. In addition, antibodies are often large immunogenicmolecules and can induce an immune response in the patient directedagainst the therapeutic agent. In addition, antibodies often do not showsufficient specificity for the target (e.g., cancer) tissue and thus areuseful in only limited therapeutic regimen.

The present disclosure provides a selective high affinity ligand (SHAL)that specifically binds to a cancer cell, said SHAL comprising a firstligand that binds to a first site on a marker for said cancer cell, saidfirst ligand linked to a second ligand that binds a second site on thesame marker or a different marker for said cancer cell wherein saidfirst site and said second site are different sites; and wherein atleast said first ligand is a ligand selected from the group consistingof BOC-4-aminomethyl-L-Phe,4[[5-(Trifluoromethyl)pyridin-2-yl]oxy]phenyl]N-phenylcarbamate,(R)-2-[4-(5-chloro-3-fluoro-2-pyridyloxy)phenoxy]propionic acid,2-(((3-chloro-5(trifluoromethyl)pyridin-2-yloxy)phenoxy)methyl)acrylates,2-(((3-chloro-5(trifluoromethyl)pyridin-2-yloxy)phenyl)methyl)acrylates,2-(((3-chloro-5(trifluoromethyl)pyridin-2-yloxy)phenyl)methyl)acrylonitriles,3-(3-chloro-4-{[5-(trifluoromethyl)-2-pyridinyl]oxy}anilino)-3-oxopropanoicacid, Sethoxydim, Clethodim, 5-(Tetradecyloxy)-2-furoic acid,2-[(2,6-Dichlorophenyl)amino]benzeneacetic acid,2-[4-(4-Chlorophenoxy)phenoxy]propanoic acid,(RS)-2-{4-[3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy}propanoicacid, (RS)-2-[4-(6-chloro-1,3-benzoxazol-2-yloxy)phenoxy]propanoic acid,(RS)-2-[4-(2,4-dichlorophenoxy)phenoxy]propanoic acid,(RS)-2-{4-[5-(trifluoromethyl)-2-pyridyloxy]phenoxy}propanoic acid,(RS)-2-[4-(6-chloroquinoxalin-2-yloxy)phenoxy]propanoic acid,(RS)-2-[4-(α, α, α-trifluoro-p-tolyloxy)phenoxy]propanoic acid,5-([4,6-Dichlorotriazin-2-yl]amino)fluorescein hydrochloride,3-[N-(4-acetylphenyl)carbomoyl]pyridine-2-Carboxylic acid,3-(2-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy}anilino)-3-oxopropanoicacid, L-ornithine-beta-alanine,2-Methyl-1-(3-morpholinopropyl)-5-phenyl-1H-pyrrole-3-carboxylic acid,Hippuric acid, Hippuryl-D-lysine, and hippuryl-L-phenylalanine.

In one embodiment, said first ligand binds a site that is different thanthe site bound by the second ligand. In another embodiment, said firstsite is a pocket on said marker. In yet another embodiment, said secondsite is a pocket on said marker.

In one embodiment, said marker is an HLA-DR cell surface antigen. Inanother embodiment, said first ligand and said second ligand bind siteswithin an epitope recognized by the Lym-1 antibody.

In any of the above embodiments, said first ligand is a small organicmolecule. In any of the above embodiments, said second ligand is a smallorganic molecule.

Suitable second ligand can be selected from Tables 1, 5, 6, 7, or 8.

In one embodiment, the SHAL is tridentate further comprising a thirdligand. Suitable second and third ligands can be independently selectedfrom the group of ligands found in Tables 1, 5, 6, 7, or 8.

In one embodiment of SHAL, said third ligand is4-(Dimethylamino)azobenzene-4′-sulfonyl-L-valine (Dv); said secondligand is 4-[4-(4-chlorobenzyl)piperazino]-3-nitrobenzenecarboxylic acid(Cb); and said first ligand is selected from the group consisting of4[[5-(Trifluoromethyl)pyridin-2-yl]oxy]phenyl]N-phenylcarbamate,(R)-2-[4-(5-chloro-3-fluoro-2-pyridyloxy)phenoxy]propionic acid,2-(((3-chloro-5(trifluoromethyl)pyridin-2-yloxy)phenoxy)methyl)acrylates,2-(((3-chloro-5(trifluoromethyl)pyridin-2-yloxy)phenyl)methyl)acrylates,2-(((3-chloro-5(trifluoromethyl)pyridin-2-yloxy)phenyl)methyl)acrylonitriles,3-(3-chloro-4-{[5-(trifluoromethyl)-2-pyridinyl]oxy}anilino)-3-oxopropanoicacid, Sethoxydim, Clethodim, 5-(Tetradecyloxy)-2-furoic acid,2-[(2,6-Dichlorophenyl)amino]benzeneacetic acid,2-[4-(4-Chlorophenoxy)phenoxy]propanoic acid,(RS)-2-{4-[3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy}propanoicacid, (RS)-2-[4-(6-chloro-1,3-benzoxazol-2-yloxy)phenoxy]propanoic acid,(RS)-2-[4-(2,4-dichlorophenoxy)phenoxy]propanoic acid,(RS)-2-{4-[5-(trifluoromethyl)-2-pyridyloxy]phenoxy}propanoic acid,(RS)-2-[4-(6-chloroquinoxalin-2-yloxy)phenoxy]propanoic acid, and(RS)-2-[4-(α, α, α-trifluoro-p-tolyloxy)phenoxy]propanoic acid.

Other examples of SHAL are further provided in Table 2.

For any SHAL of the above embodiments that have two ligands, said firstligand is joined to said second ligand by a linker selected from thegroup consisting of a PEG type linker, a peptide or peptide analoglinker, an avidin/biotin linker, a straight chain carbon linker, aheterocyclic linker, a branched carbon linker, a dendrimer, a nucleicacid linker, a sugar or carbohydrate linker, a thiol linker, an esterlinker, a linker comprising an amine, and a linker comprising acarboxyl. In one embodiment, said linker comprises polyethyleneglycol.In another embodiment, said linker comprises polyethyleneglycol and alysine.

For any SHAL of the above embodiments that have three ligands, saidsecond ligand and said third ligand are joined to each other by a linkercomprising a moiety selected from the group consisting of a PEG typelinker, a peptide linker, an avidin/biotin linker, a straight chaincarbon linker, a heterocyclic linker, a branched carbon linker, adendrimer, a nucleic acid linker, a sugar or carbohydrate linker, athiol linker, an ester linker, a linker comprising an amine, and alinker comprising a carboxyl. In one embodiment, said linker comprisespolyethyleneglycol. In another embodiment, said linker comprisespolyethyleneglycol and a lysine.

Another embodiment of the present disclosure provides a selective highaffinity ligand (SHAL) that specifically binds to a cancer cell, saidSHAL comprising: three ligands attached to each other, wherein saidfirst ligand is 4-(Dimethylamino)azobenzene-4′-sulfonyl-L-valine (Dv);said second ligand is4-[4-(4-chlorobenzyl)piperazino]-3-nitrobenzenecarboxylic acid (Cb); andsaid third ligand is selected from the group consisting of3-(2-([3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanoicacid (Ct), or an analogue thereof.

In one embodiment, said first ligand, said second ligand and said thirdligand are joined to each other by a linker comprising a moiety selectedfrom the group consisting of a PEG type linker, a peptide linker, anavidin/biotin linker, a straight chain carbon linker, a heterocycliclinker, a branched carbon linker, a dendrimer, a nucleic acid linker, asugar or carbohydrate linker, a thiol linker, an ester linker, a linkercomprising an amine, and a linker comprising a carboxyl. In one aspect,said linker comprises polyethyleneglycol. In another aspect, said linkercomprises polyethyleneglycol and a lysine.

Another embodiment of the present disclosure provides a SHAL comprisingthe structure:

wherein R¹ is selected from the group consisting of COOH, a linker, aneffector, and a linker attached to an effector.

Yet another embodiment of the present disclosure provides a SHALcomprising the structure:

wherein R² comprises an effector.

In any of the above embodiments, A SHAL can be bivalent.

In some embodiments, said SHAL is attached to a transduction peptide. Inone embodiment, said transduction peptide is selected from the groupconsisting of nuclear localization signal of SV40, the proteintransduction domain of HIV Tat protein, the integrin-binding peptide(RGD peptide), the heparin-binding domain of vitronectin (VN peptide),antennapedia protein of Drosophila, VP22, oligoarginine, lactosylatedpoly-L-lysine or other oligocation, S-G-E-H-T-N-G-P-S-K-T-S-V-R-W-V-W-D,S-M-T-T-M-E-F-G-H-S-M-I-T-P-Y-K-I-D,Q-D-G-G-T-W-H-L-V-A-Y-C-A-K-S-H-R-Y,M-S-D-P-N-M-N-P-G-T-L-G-S-S-H-I-L-W,S-P-G-N-Q-S-T-G-V-I-G-T-P-S-F-S-N-H,S-S-G-A-N-Y-F-F-N-A-I-Y-D-F-L-S-N-F, andG-T-S-R-A-N-S-Y-D-N-L-L-S-E-T-L-T-Q. In another embodiment, saidtransduction peptide is hexa-arginine.

In any of the above embodiments, said SHAL is attached to an effector.In one embodiment, said effector is selected from the group consistingof an epitope tag, an antibody, a second SHAL, a label, a cytotoxin, aliposome, a radionuclide, a drug, a prodrug, an enzyme inhibitor, aviral particle, a cytokine, and a chelate. In another embodiment, saideffector is an epitope tag selected from the group consisting of anavidin, and a biotin. In yet another embodiment, said effector is acytotoxin selected from the group consisting of a Diphtheria toxin, aPseudomonas exotoxin, a ricin, an abrin, and a thymidine kinase. Instill another embodiment, said effector is a chelate comprising a metalisotope selected from the group consisting of ⁹⁹Tc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga,⁷²As, ¹¹¹In, ^(113m)In, ⁹⁷Ru, ⁶²Cu, ⁶⁴Cu, ⁵²Fe, ^(52m)Mn, ⁵¹Cr, ¹⁸⁶Re,¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au,¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm,¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh and ¹¹¹Ag. Further in another embodiment,said effector is a chelate comprising an alpha emitter. In one aspect,said alpha emitter is bismuth 213. In another embodiment, said effectoris a chelate comprising DOTA. In another embodiment, said effector is alipid or a liposome. In a particular embodiment, said effector isselected from the group consisting of3-(2-([3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanoicacid (Ct),4[[5-(Trifluoromethyl)pyridin-2-yl]oxy]phenyl]N-phenylcarbamate,(R)-2-[4-(5-chloro-3-fluoro-2-pyridyloxy)phenoxy]propionic acid,2-(((3-chloro-5(trifluoromethyl)pyridin-2-yloxy)phenoxy)methyl)acrylates,2-(((3-chloro-5(trifluoromethyl)pyridin-2-yloxy)phenyl)methyl)acrylates,2-(((3-chloro-5(trifluoromethyl)pyridin-2-yloxy)phenyl)methyl)acrylonitriles,3-(3-chloro-4-{[5-(trifluoromethyl)-2-pyridinyl]oxy}anilino)-3-oxopropanoicacid, Sethoxydim, Clethodim, 5-(Tetradecyloxy)-2-furoic acid,2-[(2,6-Dichlorophenyl)amino]benzeneacetic acid,2-[4-(4-Chlorophenoxy)phenoxy]propanoic acid,(RS)-2-{4-[3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy}propanoicacid, (RS)-2-[4-(6-chloro-1,3-benzoxazol-2-yloxy)phenoxy]propanoic acid,(RS)-2-[4-(2,4-dichlorophenoxy)phenoxy]propanoic acid,(RS)-2-{4-[5-(trifluoromethyl)-2-pyridyloxy]phenoxy}propanoic acid,(RS)-2-[4-(6-chloroquinoxalin-2-yloxy)phenoxy]propanoic acid, and(RS)-2-[4-(α, α, α-trifluoro-p-tolyloxy)phenoxy]propanoic acid.

One embodiment of the present disclosure provides a method of inhibitingthe growth or proliferation of a cancer cell that expresses an HLA-DR10marker, said method comprising: contacting said cancer with a selectivehigh-affinity polydentate ligand (SHAL) of any of the above embodiments.

In one aspect, said cell is a metastatic cell. In another aspect, saidcell is a solid tumor cell. In yet another aspect, said cancer cell is amalignant B lymphocyte. In a particular aspect, said cancer cell isassociated with non-Hodgkins lymphoma or leukemia or other B-cellderived malignancies.

Still in another embodiment, the present disclosure provides apharmaceutical formulation said formulation comprising: apharmaceutically acceptable excipient and a SHAL of any of the aboveembodiments. In one aspect, said formulation is a unit dosageformulation.

Further provided, in one embodiment, is a method of detecting a cancercell, said method comprising: contacting said cancer cell with achimeric molecule comprising an SHAL of any of the above embodimentsattached to a detectable label; and detecting the presence or absence ofsaid detectable label. In one aspect, said detectable label is aselected from the group consisting of a gamma-emitter, apositron-emitter, an x-ray emitter, an alpha emitter, and afluorescence-emitter.

Still further provided, in one embodiment, is a method of detecting acancer cell, said method comprising: contacting a cancer cell with achimeric molecule comprising chimeric molecule comprising a SHAL of anyof the above embodiments attached to an epitope tag; contacting saidchimeric molecule with a chelate comprising a detectable moiety wherebysaid chelate binds to said epitope tag thereby associating saiddetectable moiety with said chelate; and detecting said detectablemoiety.

In one aspect, said detectable moiety is a radionuclide. In anotheraspect, said detectable moiety is selected from the group consisting ofa gamma-emitter, a positron-emitter, an alpha emitter, an x-ray emitter,and a fluorescence-emitter. In one aspect, said detecting comprisesexternal imaging. In another aspect, said detecting comprises internalimaging.

In a particular aspect, said detectable moiety comprises a metal isotopeselected from the group consisting of ⁹⁹Tc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As,¹¹¹In, ^(113m)In, ⁹⁷Ru, ⁶²Cu, ^(64l)Cu, ⁵²Fe, ^(52m)Mn, ⁵¹Cr, ¹⁸⁶Re,¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au,¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm,¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh, and ¹¹¹Ag. In another aspect, said chelatecomprises DOTA. In yet another aspect, said epitope tag is an avidin, abiotin, or an enzyme.

SUMMARY

In certain embodiments this invention provides novel polydentateselective high affinity ligands (SHALs) that can function tospecifically bind particular target molecules in a manner analogous toantibody binding. Methods for the design and generation of SHALs asaffinity reagents for basic or applied research or for diagnosis andtreatment of infectious and/or malignant diseases and theiradministration to patients with infectious and/or malignant diseases aredescribed.

The SHALs typically comprise two or more ligands (binding moieties) thateach bind different regions on the intended target attached to eachother directly or through a linker. Where the SHAL is directed to amarker on a cancer cell, the SHAL associates in greater density(abundance) or accessibility on the target cell as compared to normalcells. The SHAL thus provides selectivity appropriate for diagnosis ortreatment of the target cells. Different SHALs can be readily generatedfor different malignant cells and malignant diseases. The SHALrepresents a core building block (e.g., a targeting moiety) that can beincorporated into larger e.g., chimeric molecules to affect specificdelivery of an effector to the target.

Thus, in one embodiment, this invention provides a method of making aselective high-affinity polydentate ligand (SHAL) that specificallybinds a target molecule. In certain embodiments, the method typicallyinvolves screening a first ligand library to identify a first ligandthat binds to the target molecule; screening a second ligand library toidentify a second ligand that binds to the target molecule where thesecond ligand is different than the first ligand; linking the firstligand to the second ligand to form a SHAL; and screening the SHAL forthe ability to specifically bind to the target molecule. In certainembodiments the target molecule is a protein. In certain embodiments thetarget molecule is a cancer marker (e.g., Lym-1 epitope, Muc-1, C-myc,p53, Ki67, Her2, Her4, BRCA1, BRCA2, Lewis Y, CA 15-3, G250, HLA-DR cellsurface antigen, CEA, CD20, CD22, integrin, cea, l6, EGFr, AR, PSA, andother growth factor receptors, etc.). The method can optionally furtherinvolve screening the SHAL to identify a SHAL that binds to the targetwith an avidity and/or specificity higher than either ligand comprisingthe SHAL. The first ligand library and the second ligand library can bethe same library or can be different libraries. In certain embodimentsthe first and/or second ligand library is a library of small organicmolecules. In certain embodiments screening the first ligand libraryand/or screening the second ligand library comprises virtual in silicoscreening. The virtual in silico screening can comprise screening acompound database (e.g., MDL® Available Chemicals Directory, ChemSpider,GNU-Darwin) using one or more algorithms as utilized in the DOCKprogram. The virtual in silico screening can comprise screening acompound database using the DOCK program. The virtual in silicoscreening can involve screening for a first ligand and/or multipleligands that bind a pocket on a protein. In certain embodiments thepocket is identified using an algorithm utilized by the SPHGEN program.In certain embodiments the pocket is identified using the SPHGENprogram. The virtual in silico screening can involve screening for asecond or third ligand that binds different regions of the target thanthe ligands identified when screening the first ligand library.

In certain embodiments screening a first ligand library and/or screeninga second ligand library additionally comprises screening one or moreligands identified in the virtual in silico screening in a physicalassay for the ability to bind to the target. Suitable physical assaysinclude, but are not limited to a BIAcore assay, saturation transferdifference nuclear magnetic resonance spectroscopy, and transfer NOE(trNOE) nuclear magnetic resonance spectroscopy, ELISA, competitiveassay, tissue binding assay, a live cell binding assay, a cellularextract assay, and the like.

Linking of the ligands can involve directly linking two or more ligandsor linking two or more ligands with a linker (e.g., a PEG type linker, apeptide linker, an avidin/biotin linker, a straight chain carbon linker,a heterocyclic linker, a branched carbon linker, a dendrimer, a nucleicacid linker, a thiol linker, an ester linker, a linker comprising anamine, a linker comprising a carboxyl, etc.). The linking can optionallycomprise linking two or more ligands with linkers of different lengthsto produce a library of SHALs having different length linkers; and,optionally, screening the library of SHALs having different lengthlinkers to identify members of the library that have the highest avidityand/or specificity for the target. In certain embodiments the methodfurther involves comprising screening the SHAL(s) to identify a SHALthat binds to the target with an avidity and/or specificity higher thaneither ligand comprising the SHAL. The screening of individual ligandsand/or bivalent or polyvalent SHAL(s) can be by any of a variety ofmethods including, but not limited to a BIAcore assay, saturationtransfer difference nuclear magnetic resonance spectroscopy, andtransfer NOE (trNOE) nuclear magnetic resonance spectroscopy, ELISA,competitive assay, tissue binding assay, live cell binding assay, acellular extract assay, and the like. In certain embodiments the targetmolecule is a protein and/or a cancer marker (e.g., a Lym1 epitope,Muc-1, C-myc, p53, Ki67, Her2, Her4, BRCA1, BRCA2, Lewis Y, CA 15-3,G250, HLA-DR cell surface antigen, etc.).

Also provided is a method of synthesizing an inhibitor for an enzyme orother binding protein or receptor. In certain embodiments the methodtypically involves identifying a first pocket (or bump) and a second orthird pocket (or bump) in the enzyme or other binding protein orreceptor where the first, second and third pockets flank opposite sidesof the active site or binding site of the enzyme or other bindingprotein or receptor; screening a first ligand library to identify afirst ligand that binds to the first pocket (or bump); screening asecond ligand library to identify a second ligand that binds to thesecond pocket (or bump); screening a third ligand library to identify athird ligand that binds to a third pocket or bump; linking the firstligand to the second and third ligands to form a polydentate selectivehigh affinity ligand (SHAL); and screening the SHAL for the ability tospecifically bind to and inhibit the enzyme or other binding protein. Incertain embodiments the pockets or “bumps” need not be located onopposite sides of the active site or binding site of the enzyme orbinding protein or receptor, but are simply located so that binding ofthe SHAL blocks binding of the native cognate ligand to that site. Incertain embodiments the target molecule comprises a molecule selectedfrom the group consisting of a protein, an enzyme, a nucleic acid, anucleic acid binding protein, and a carbohydrate. In certain embodimentsthe method further involves screening the SHAL to identify a SHAL thatbinds to the target with an avidity and/or specificity higher thaneither ligand comprising the SHAL. The first, second and third ligandlibraries can be the same library or can be different libraries. Incertain embodiments the first and/or second and/or third ligand libraryis a library of small organic molecules. In certain embodimentsscreening the first, second and/or third ligand library comprisesvirtual in silico screening. The virtual in silico screening cancomprise screening a compound database (e.g., MDL® Available ChemicalsDirectory) using one or more algorithms as utilized in the DOCK program.The virtual in silico screening can comprise screening a compounddatabase using the DOCK program. The virtual in silico screening caninvolve screening for a first, second and/or third ligand that binds apocket on a protein. In certain embodiments the pocket is identifiedusing an algorithm utilized by the SPHGEN program. In certainembodiments the pocket is identified using the SPHGEN program. Thevirtual in silico screening can involve screening for a second ligandthat binds different region of the target than the ligands identifiedwhen screening the first ligand library.

In certain embodiments screening a first, second or third ligand libraryand/or screening a second ligand library additionally comprisesscreening one or more ligands identified in the virtual in silicoscreening in a physical assay for the ability to bind to the target.Suitable physical assays include, but are not limited to a BIAcoreassay, saturation transfer difference nuclear magnetic resonancespectroscopy, and transfer NOE (trNOE) nuclear magnetic resonancespectroscopy, ELISA, competitive assay, tissue binding assay, a livecell binding assay, a cellular extract assay, and the like.

Linking of the ligands can involve directly linking two or more ligandsor linking two or more ligands with a linker (e.g., a PEG type linker, apeptide linker, an avidin/biotin linker, a straight chain carbon linker,a heterocyclic linker, a branched carbon linker, a dendrimer, a nucleicacid linker, a thiol linker, an ester linker, a linker comprising anamine, a linker comprising a carboxyl, etc.). The linking can optionallycomprise linking the ligands with linkers of different lengths toproduce a library of SHALs having different length linkers; and,optionally, screening the library of SHALs having different lengthlinkers to identify members of the library that have the highest avidityand/or specificity for the target.

This invention also provides a polydentate selective high affinityligand (SHAL) that specifically binds to a desired target (e.g., acancer cell). Where the target is a cancer cell, the SHAL typicallycomprises a first ligand that binds to a first site on a marker for thecancer cell linked (directly or through a linker) to a second or thirdligand that binds to a second or third site on same marker or on adifferent marker for the cancer cell where the first site, the secondsite and the third site are different sites (e.g., all three ligands arecapable of simultaneously binding to the target(s)). In certainembodiments the first site, second site and/or the third site is apocket (or “bump”) on the marker(s). Suitable markers include, but arenot limited to a Lym-1 epitope, Muc-1, C-myc, p53, Ki67, Her2, Her4,BRCA1, BRCA2, Lewis Y, CA 15-3, G250, HLA-DR cell surface antigen, CEA,CD20, CD22, integrin, CEA, l6, EGFr, AR, PSA, other growth factorreceptors, and the like.

In certain preferred embodiments, the marker is an HLA-DR cell surfaceantigen. In certain embodiments the three ligands bind sites within anepitope recognized by the Lym-1 or other HLA-DR specific antibodies. Incertain embodiments the three ligands are each small organic molecules.In certain embodiments, the first ligand is a ligand selected from Table1, and the second and third ligand, when present, are independentlyselected from the ligands in Tables 1, 5, 6, 7, or 8. In certainembodiments the SHAL comprises a first, second and/or third ligandselected from Tables 2, 3, or 4. In certain embodiments the SHALcomprises a first, second and/or third ligand selected from Table 4. Thethree ligands can be joined directly together or the first ligand can beattached to the second and/or third ligand by a linker (e.g., a PEG typelinker, a peptide linker, an avidin/biotin linker, a straight chaincarbon linker, a heterocyclic linker, a branched carbon linker, adendrimer, a nucleic acid linker, a thiol linker, an ester linker, alinker comprising an amine, a linker comprising a carboxyl, etc.). Incertain embodiments the SHAL has an avidity for the marker greater thanabout 10⁻⁶ M while the individual ligands comprising the SHAL each havea binding affinity for the marker less than about 10⁻⁶ M. In certainembodiments the SHAL has a formula as shown herein and in the Figures oris an analogue thereof.

This invention also provides chimeric molecules comprising a SHAL asdescribed herein attached to an effector (e.g., an epitope tag, a secondSHAL, an antibody, a label, a cytotoxin, a liposome, a radionuclide, adrug, a prodrug, a viral particle, a cytokine, and a chelate. In certainembodiments the effector is an epitope tag selected from the groupconsisting of an avidin, and a biotin. In certain embodiments theeffector is a cytotoxin selected from the group consisting of aDiphtheria toxin, a Pseudomonas exotoxin, a ricin, an abrin, and athymidine kinase. In certain embodiments the effector is a chelatecomprising a metal isotope selected from the group consisting of ⁹⁹Tc,²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ^(113m)In, ⁹⁷Ru, ⁶²Cu, ⁶⁴Cu, ⁵²Fe,^(52m)Mn, ⁵¹Cr, ¹⁸⁶, Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ¹²¹Sn, ¹²⁷Te,¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm,¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh, and ¹¹¹Ag. Incertain embodiments effector is a chelate comprising an alpha emitter(e.g., bismuth 213). In certain embodiments the effector is a chelatecomprising DOTA. In certain embodiments the effector is a lipid or aliposome (e.g., a liposome containing a drug).

In still another embodiment, this invention provides a pharmaceuticalformulation the formulation comprising a polydentate selective highaffinity ligand (SHAL) that specifically binds to a cancer cell asdescribed herein and a pharmaceutically acceptable excipient. In certainembodiments the formulation can be provided as a unit dosageformulation. In certain embodiments the formulation can be provided as atime-release formulation.

This invention also provides a pharmaceutical formulation theformulation comprising a pharmaceutically acceptable excipient and achimeric molecule comprising a SHAL as described herein. In certainembodiments the formulation can be provided as a unit dosageformulation. In certain embodiments the formulation can be provided as atime-release formulation.

Methods are provided for inhibiting the growth or proliferation of acancer cell. The methods typically involves contacting the cancer cell(e.g., metastatic cell, tumor cell, etc.) with a polydentate selectivehigh affinity ligand (SHAL) that specifically binds to a cancer celland/or with a chimeric molecule comprising a polydentate selective highaffinity ligand (SHAL) that specifically binds to a cancer cell attachedto an effector (e.g., drug, liposome, cytotoxin, radionuclide, orchelator).

In certain embodiments, this invention provides SHALS that specificallybind to a desired target. The target can be any target for which it isdesired to create a binding moiety. The SHAL typically comprises two ormore ligands joined directly or through a linker where a first ligandthat binds to a first site on the target and the second and/or thirdligand binds to second and/or third site on the target on same targetmarker where the first, second and/or third sites are different sites(e.g., all three ligands are capable of simultaneously binding to thetarget(s)). In certain embodiments the first, second and/or third siteis a pocket (or “bump”) on the target(s). In certain embodiments thefirst, second and/or third sites are on the same target molecule.

This invention also provides various detection methods. In certainembodiments this invention provides a method of detecting a cancer cell.The method typically involves contacting the cancer cell with a chimericmolecule comprising a SHAL that specifically binds to a cancer cell(e.g., to a cancer marker) attached to a detectable label (e.g.,gamma-emitter, a positron-emitter, an x-ray emitter, an alpha emitter, afluorescence-emitter, etc.) and detecting the presence or absence of thedetectable label. In certain embodiments the method typically involvescontacting a cancer cell with a chimeric molecule comprising chimericmolecule comprising SHAL that specifically binds to a cancer cell (e.g.,to a cancer marker) attached to an epitope tag; contacting the chimericmolecule with a chelate comprising a detectable moiety whereby thechelate binds to the epitope tag thereby associating the detectablemoiety with the chelate; and detecting the detectable moiety. In certainembodiments the detectable moiety is a radionuclide (e.g., agamma-emitter, a positron-emitter, an alpha emitter, an x-ray emitter,etc.). In certain embodiments the detecting comprises external imagingand/or internal imaging. In certain embodiments the detectable moietycomprises a metal isotope selected from the group consisting of ⁹⁹Tc,²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ^(113m)In, ⁹⁷Ru, ⁶²Cu, ^(64l)Cu, ⁵²Fe,^(52m)Mn, ⁵¹Cr, ¹⁸⁶, Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ¹²¹Sn, ¹²⁷Te,¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm,¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh, and ¹¹¹Ag. Incertain embodiments the chelate comprises DOTA. In certain embodimentsthe epitope tag is an avidin or a biotin.

This invention also contemplates kits for creating and/or using SHALs ofthis invention and/or chimeric molecules comprising SHALs of thisinvention. In certain embodiments the kit comprises a containercontaining a SHAL as described herein and/or containers containingligands for assembly into a SHAL as described herein. The like canoptionally further include one or more linkers, one or more effectors(chelates, radionuclides, etc.), and the like. In certain embodimentsthe SHAL is in a pharmacologically acceptable excipient.

In certain embodiments, this invention expressly excludes SHALs wherethe binding moieties comprising the SHAL are antibodies, single chainantibodies, and the like. In certain embodiments the SHALs are notpolyvalent antibodies or polyvalent single chain antibodies. In certainembodiments the ligands comprising the SHALs are not proteins. Incertain embodiments, this invention expressly excludes SHALS where thebinding moieties comprising the SHAL preferentially and/or specificallybind nucleic acids. In certain embodiments the ligands comprising theSHALs are small organic molecules.

Definitions

The terms “specific binding” or “preferential binding” refer to thatbinding which occurs between such paired species as enzyme/substrate,receptor/agonist, antibody/antigen, and lectin/carbohydrate which may bemediated by covalent and/or non-covalent interactions. When theinteraction of the two species typically produces a non-covalently boundcomplex, the binding which occurs is typically electrostatic, and/orhydrogen-bonding, and/or the result of lipophilic interactions.Accordingly, “specific binding” occurs between pairs of species wherethere is interaction between the two that produces a bound complex. Inparticular, the specific binding is characterized by the preferentialbinding of one member of a pair to a particular species as compared tothe binding of that member of the pair to other species within thefamily of compounds to which that species belongs. Thus, for example, aligand may show an affinity for a particular pocket on a HLA-DR10molecule that is at least two-fold preferably at least 10 fold, morepreferably at least 100 fold, at least 1000 fold, or at least 10000 foldgreater than its affinity for a different pocket on the same or relatedproteins.

The terms “ligand” or “binding moiety”, as used herein, refers generallyto a molecule that binds to a particular target molecule and forms abound complex as described above. The binding can be highly specificbinding, however, in certain embodiments, the binding of an individualligand to the target molecule can be with relatively low affinity and/orspecificity. The ligand and its corresponding target molecule form aspecific binding pair. Examples include, but are not limited to smallorganic molecules, sugars, lectins, nucleic acids, proteins, antibodies,cytokines, receptor proteins, growth factors, nucleic acid bindingproteins and the like which specifically bind desired target molecules,target collections of molecules, target receptors, target cells, and thelike.

The term “small organic molecule” refers to a molecule of a sizecomparable to those organic molecules generally used in pharmaceuticals.The term excludes natural biological macromolecules (e.g., proteins,nucleic acids, etc.). Preferred small organic molecules range in size upto about 5000 Da, more preferably up to 2000 Da, and most preferably upto about 1000 Da.

The term “ligand library” refers to a collection (e.g., to a plurality)of ligands or potential ligands. The ligand library can be an actualphysical library of ligands and/or a database (e.g., a compound databasecomprising descriptions of a plurality of potential ligands such as theMDL® Available Chemicals Directory, ChemSpider, and the like).

The term “SHAL” refers to a molecule comprising a plurality of ligandsthat each bind to a different region of the target molecule to which theSHAL is directed. The ligands are joined together either directly orthrough a linker to form a polydentate moiety that typically shows highavidity for the target molecule. In certain embodiments, the SHALcomprises two or more ligands that bind their target with low affinity(e.g., <10⁻⁶M and/or dissociates within seconds or less) that, whencoupled together, form a SHAL that binds the target with high affinity(e.g., >10⁻⁶M, or >10⁻⁷M, or >10⁻⁸ M and/or dissociates slowly, e.g.,hours to days).

The term “polydentate” when used with respect to a SHAL indicates thatthe SHAL comprises two or more ligands. The ligands typically bind todifferent parts of the target to which the SHAL is directed.

The terms “bidentate”, “tridentate”, and so forth when used with respectto a SHAL refer to SHALs consisting of two ligands, SHALs consisting ofthree ligands, and so forth.

The term “polyvalent SHAL” refers to a molecule in which two or moreSHALs (e.g., two or more bidentate SHALs) are joined together. Thus, forexample a bivalent SHAL refers to a molecule in which two SHALs arejoined together. A trivalent SHAL refers to a molecule in which threeSHALs are joined together, and so forth. A bivalent version of thebidentate SHAL JP459B is illustrated in FIG. 14).

A “polyspecific SHAL” is 2 or more SHALs joined together where each SHALis polydentate and either or both can be polyvalent synthesized (orotherwise generated) so that they have 2 or more targets for each SHAL(set of poly ligands). For example, a SHAL can be synthesized with twoor more ligands for the cavities of HLA-DR and cavities on a CDXX, egCD20 or CD22, or all 3, etc. Another example involves joining a MUC-1SHAL and an antilyphoma SHAL because some lymphomas overexpresstraditional HLA-DR and CD receptors and MUC-1 (upregulated). SHALsynthesized with 2 or more ligands for the cavities of HLA-DR andcavities for a chelate, e.g DOTA, etc. where in the univalent orbivalent SHAL targets the malignant cell and the univalent or bivalent2nd module catches a subsequently delivered agent, eg DOTA chelatedradiometal or a prodrug intended to activate the drug transported to themalignant cell by the 1st SHAL.

The term “virtual in silico” when used, e.g., with respect to screeningmethods refers to methods that are performed without actual physicalscreening of the subject moieties. Typically virtual in silico screeningis accomplished computationally, e.g., utilizing models of theparticular molecules of interest. In certain embodiments, the virtualmethods can be performed using physical models of the subject moleculesand/or by simple visual inspection and manipulation.

The phrase “target for a SHAL” refers to the moiety that is to bespecifically bound by the bidentate or polydentate SHAL.

The phrase “an algorithm found in . . . ”, e.g., “an algorithm found inSPHGEN” refers to an algorithm that is implemented by (found in) thereferenced software. The algorithm, however, can be manually, or by aprogram other than the referenced software and still represent a use ofan algorithm found in the referenced software.

The term “pocket” when referring to a pocket in a protein refers to acavity, indentation or depression in the surface of the protein moleculethat is created as a result of the folding of the peptide chain into the3-dimensional structure that makes the protein functional. A pocket canreadily be recognized by inspection of the protein structure and/or byusing commercially available modeling software (e.g., DOCK).

The term “cancer markers” refers to biomolecules such as proteins thatare useful in the diagnosis and prognosis of cancer. As used herein,“cancer markers” include but are not limited to: PSA, human chorionicgonadotropin, alpha-fetoprotein, carcinoembryonic antigen, cancerantigen (CA) 125, CA 15-3, CD20, CDH13, CD 31, CD34, CD105, CD146,D16S422HER-2, phosphatidylinositol 3-kinase (PI 3-kinase), trypsin,trypsin-1 complexed with alpha(1)-antitrypsin, estrogen receptor,progesterone receptor, c-erbB-2, bc1-2, S-phase fraction (SPF),p185erbB-2, low-affinity insulin like growth factor-binding protein,urinary tissue factor, vascular endothelial growth factor, epidermalgrowth factor, epidermal growth factor receptor, apoptosis proteins(p53, Ki67), factor VIII, adhesion proteins (CD-44, sialyl-TN, bloodgroup A, bacterial lacZ, human placental alkaline phosphatase (ALP),alpha-difluoromethylornithine (DFMO), thymidine phosphorylase(dTHdPase), thrombomodulin, laminin receptor, fibronectin, anticyclins,anticyclin A, B, or E, proliferation associated nuclear antigen, lectinUEA-1, CEA, l6, and von Willebrand's factor.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The term also includes variants on the traditional peptidelinkage joining the amino acids making up the polypeptide.

The terms “nucleic acid” or “oligonucleotide” or grammatical equivalentsherein refer to at least two nucleotides covalently linked together. Anucleic acid of the present invention is preferably single-stranded ordouble stranded and will generally contain phosphodiester bonds,although in some cases, as outlined below, nucleic acid analogs areincluded that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925) andreferences therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl etal. (1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986) Nucl. AcidsRes. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al.(1988) J. Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986) ChemicaScripta 26: 1419), phosphorothioate (Mag et al. (1991) Nucleic AcidsRes. 19:1437; and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu etal. (1989) J. Am. Chem. Soc. 111:2321, O-methylphophoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al.(1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen (1993) Nature, 365: 566;Carlsson et al. (1996) Nature 380: 207). Other analog nucleic acidsinclude those with positive backbones (Denpcy et al. (1995) Proc. Natl.Acad. Sci. USA 92: 6097; non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew. (1991) Chem. Intl.Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470;Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and3, ASC Symposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al. (1994),Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J.Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Ed. Y. S. Sanghui and P. Dan Cook.Nucleic acids containing one or more carbocyclic sugars are alsoincluded within the definition of nucleic acids (see Jenkins et al.(1995), Chem. Soc. Rev. pp 169-176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. These modificationsof the ribose-phosphate backbone may be done to facilitate the additionof additional moieties such as labels, or to increase the stability andhalf-life of such molecules in physiological environments.

The term “biotin” refers to biotin and modified biotins or biotinanalogues that are capable of binding avidin or various avidinanalogues. “Biotin”, can be, inter alia, modified by the addition of oneor more addends, usually through its free carboxyl residue. Usefulbiotin derivatives include, but are not limited to, active esters,amines, hydrazides and thiol groups that are coupled with acomplimentary reactive group such as an amine, an acyl or alkyl group, acarbonyl group, an alkyl halide or a Michael-type acceptor on theappended compound or polymer.

Avidin, typically found in egg whites, has a very high binding affinityfor biotin, which is a B-complex vitamin (Wilcheck et al. (1988) Anal.Biochem, 171: 1). Streptavidin, derived from Streptomyces avidinii, issimilar to avidin, but has lower non-specific tissue binding, andtherefore often is used in place of avidin. As used herein “avidin”includes all of its biological forms either in their natural states orin their modified forms. Modified forms of avidin which have beentreated to remove the protein's carbohydrate residues (“deglycosylatedavidin”), and/or its highly basic charge (“neutral avidin”), forexample, also are useful in the invention.

The term “residue” as used herein refers to natural, synthetic, ormodified amino acids.

As used herein, an “antibody” refers to a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as myriad immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H)1 by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)₂ dimer into aFab′ monomer. The Fab′ monomer is essentially a Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Preferred antibodies include single chainantibodies (antibodies that exist as a single polypeptide chain), morepreferably single chain Fv antibodies (sFv or scFv) in which a variableheavy and a variable light chain are joined together (directly orthrough a peptide linker) to form a continuous polypeptide. The singlechain Fv antibody is a covalently linked V_(H)-V_(L) heterodimer whichmay be expressed from a nucleic acid including V_(H)- and V_(L)-encodingsequences either joined directly or joined by a peptide-encoding linker.Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. Whilethe V_(H) and V_(L) are connected to each as a single polypeptide chain,the V_(H) and V_(L) domains associate non-covalently. The firstfunctional antibody molecules to be expressed on the surface offilamentous phage were single-chain Fv's (scFv), however, alternativeexpression strategies have also been successful. For example Fabmolecules can be displayed on phage if one of the chains (heavy orlight) is fused to g3 capsid protein and the complementary chainexported to the periplasm as a soluble molecule. The two chains can beencoded on the same or on different replicons; the important point isthat the two antibody chains in each Fab molecule assemblepost-translationally and the dimer is incorporated into the phageparticle via linkage of one of the chains to, e.g., g3p (see, e.g., U.S.Pat. No. 5,733,743). The scFv antibodies and a number of otherstructures converting the naturally aggregated, but chemically separatedlight and heavy polypeptide chains from an antibody V region into amolecule that folds into a three dimensional structure substantiallysimilar to the structure of an antigen-binding site are known to thoseof skill in the art (see e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and4,956,778). Particularly preferred antibodies should include all thathave been displayed on phage or yeast (e.g., scFv, Fv, Fab and disulfidelinked Fv (Reiter et al. (1995) Protein Eng. 8: 1323-1331).

The term “specifically binds”, as used herein, when referring to a SHALor to a biomolecule (e.g., protein, nucleic acid, antibody, etc.),refers to a binding reaction that is determinative of the presence ofthe SHAL or biomolecule in a heterogeneous population of molecules(e.g., proteins and other biologics). Thus, under designated conditions(e.g., binding assay conditions in the case of a SHAL or stringenthybridization conditions in the case of a nucleic acid), the specifiedligand or SHAL preferentially binds to its particular “target” moleculeand preferentially does not bind in a significant amount to othermolecules present in the sample.

An “effector” refers to any molecule or combination of molecules whoseactivity it is desired to deliver/into and/or localize at a target(e.g., at a cell displaying a characteristic marker). Effectors include,but are not limited to labels, cytotoxins, enzymes, growth factors,transcription factors, drugs, lipids, liposomes, etc.

A “reporter” is an effector that provides a detectable signal (e.g., isa detectable label). In certain embodiments, the reporter need notprovide the detectable signal itself, but can simply provide a moietythat subsequently can bind to a detectable label.

The term “conservative substitution” is used in reference to proteins orpeptides to reflect amino acid substitutions that do not substantiallyalter the activity (specificity or binding affinity) of the molecule.Typically, conservative amino acid substitutions involve substitution ofone amino acid for another amino acid with similar chemical properties(e.g., charge or hydrophobicity). The following six groups each containamino acids that are typical conservative substitutions for oneanother: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid(D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine(R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine(V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The terms “epitope tag” or “affinity tag” are used interchangeablyherein, and usually refers to a molecule or domain of a molecule that isspecifically recognized by an antibody or other binding partner. Theterm also refers to the binding partner complex as well. Thus, forexample, biotin or a biotin/avidin complex are both regarded as anaffinity tag. In addition to epitopes recognized in epitope/antibodyinteractions, affinity tags also comprise “epitopes” recognized by otherbinding molecules (e.g., ligands bound by receptors), ligands bound byother ligands to form heterodimers or homodimers, His₆ bound by Ni-NTA,biotin bound by avidin, streptavidin, or anti-biotin antibodies, and thelike.

Epitope tags are well known to those of skill in the art. Moreover,antibodies specific to a wide variety of epitope tags are commerciallyavailable. These include but are not limited to antibodies against theDYKDDDDK (SEQ ID NO:1) epitope, c-myc antibodies (available from Sigma,St. Louis), the HNK-1 carbohydrate epitope, the HA epitope, the HSVepitope, the His₄, His₅, and His₆ epitopes that are recognized by theHis epitope specific antibodies (see, e.g., Qiagen), and the like. Inaddition, vectors for epitope tagging proteins are commerciallyavailable. Thus, for example, the pCMV-Tag1 vector is an epitope taggingvector designed for gene expression in mammalian cells. A target geneinserted into the pCMV-Tag1 vector can be tagged with the FLAG® epitope(N-terminal, C-terminal or internal tagging), the c-myc epitope(C-terminal) or both the FLAG (N-terminal) and c-myc (C-terminal)epitopes.

A PEG type linker refers to a linker comprising a polyethylene glycol(PEG).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a method of creating SHALs by identifying individualligands (blue) that bind to two unique sites on the surface of a target(e.g., HLA-DR10 encompassing the Lym-1 epitope (red, green, yellow aminoacids)) and linking them together synthetically to produce a moleculethat binds to both sites.

FIG. 2 illustrates the use of tissue microarrays that contain, forexample, a large number of both normal tissues and lymphocyticneoplasms. These tissues can be treated with biotin-tagged SHALs, rinsedwith rhodamine-tagged streptavidin, and the binding of the SHAL assessedby fluorescence microscopy.

FIG. 3 illustrates a human tissue microarray comprising hematoxylin andeosin stained, formalin-fixed, paraffin embedded tissue cores. All fourpanels are derived from a single ScanScope digital image. Thisillustrates the high resolution of the archived image. Similar imagescan be captured for immunohistochemical or SHAL binding studies. In thisway, investigators using the arrays have easy shared access for allstudies applied to the arrays.

FIG. 4A illustrates the organization of normal MUC-1. Abnormal MUC1,also a good target, is less glycosylated, has an exposed VNTR, and atandem repeat unit: 20 aa GVTSAPDTRPAPGSTAPPAH (SEQ ID NO:2). FIG. 4Billustrates the conserved structural features of the random repeatdomain of MUC-1 (SEQ ID NO:30). These features include repeating andprotruding knob-like structures consisting of sequential reverse turnsthat span tandem repeat interfaces (residues 17-27, 37-47), an extendedregion consisting of polyproline II and β-strand structure (residues10-15, 30-35, 50-55). The N- and C-terminal 2-3 residues are unordereddue to the absence of adjoining tandem repeats.

FIG. 5 illustrates a prostate cancer tissue array stained by anti-muc1monoclonal antibodies.

FIG. 6 shows that prostate cancer had increased MUC-1 peptide stainingassociated with grade and stage.

FIG. 7 shows anti MUC1 peptide core MoAb BrE-3 demonstrating targetepitope in grade 4 prostate cancer. The knobs on MUC-1 differ indifferent grades of prostate cancer. Consequently, SHALS can be designedthat bind to knobs and that can be used to grade malignant tissue.

FIG. 8 illustrates the amino acid sequence alignment of HLA-DR moleculeswith known crystal structures. PDB codes identify the HLA-DR1(1aqd),HLA-DR2(1bx2), HLA-DR3(1a6a) and HLA-DR4 molecules (1dm5) sequences.1i3r is a homologous MHC fusion protein. Hla_DR10: (SEQ ID NO:3),1aqd_B: (SEQ ID NO:4), 1d5m_B (SEQ ID NO:5), 1bx2_B: (SEQ ID NO:6),1a6a_B: (SEQ ID NO:7), 1i3r_B: (SEQ ID NO:8).

FIG. 9 Homology model of beta subunit of HLA-DR10 showing the twostructural domains (See Appendix for color).

FIG. 10 shows superposition of HLA-DR3 crystal structure (light blue,transparent atoms) and homology model of HLA-DR10 (dark blue, solidatoms) showing the structural similarity in the region of the betasubunit that comprises the Lym-1 epitope. The amino acid residuescritical for Lym-1 binding are shown as space filling atoms.

FIG. 11 shows surface plots of HLA-DR10 and HLA-DR3 beta subunitsshowing differences in the structure of the “pockets” in the region ofthe protein that comprises the Lym-1 epitope. Charge distribution isshown as dark blue (positive or basic), red (negative or acidic) andlight blue (neutral or hydrophobic). The sulfur atom in cysteine isshown in yellow.

FIG. 12 illustrates the location of two “pockets”, designated Site 1(red) and Site 2 (blue), that surround the amino acids critical forLym-1 binding (yellow) on the (3-subunit of HLA-DR10. These two siteswere targeted for ligand binding and used in the computational dockingstudies.

FIGS. 13A and 13B illustrate bidentate SHALs synthesized by combiningthe appropriate pairs of the individual ligands identified to bind toHLA-DR10. FIG. 13A illustrates the three molecules. FIG. 13B illustratestwo SHALs, synthesized by linking together deoxycholate and5-leu-enkephalin, that have been shown to bind to isolated HLA-DR10 withnM affinities. The red part is one ligand (deoxycholate), the green isthe other ligand (e.g, 5-leu-enkephalin), the blue is a lysine used tomake the shortest linker, and the black is a combination of things: thePEG molecules used to make the linker between the two ligands longer,and a biotin molecule attached to the SHALs for testing purposes.

FIG. 14 illustrates the structure of a bivalent version of JP459B. Redis deoxycholate; Green is 5-leu-enkephalin; Blue is the lysine linker;and Black is the PEG linker and biotin.

FIG. 15 illustrates a flowchart describing major steps in homology-basedprotein modeling.

FIG. 16 illustrates a DOCK algorithm. (1) A “negative image” isgenerated by filling a pocket with spheres. (2) A candidate ligand isretrieved from a database. (3) Internal distances are matched between asubset of sphere centers and ligand atoms (usually three to eightcenters are chosen). (4) The ligand is oriented into the active site.(5) The interaction for that orientation is evaluated by a scoringfunction; the process is repeated for new orientations—typically 10,000orientations are generated per ligand. The top-scoring orientation isretained. The process is repeated for a new ligand in the database

FIGS. 17A and 17B illustrate binding of SHAL JP459B to cells displayingHLA-DR10. FIG. 17A illustrates increased binding of SHAL JP459B andLym-1 MAb on large cell lymphoma compared to small cell lymphoma (panelsA-D) and selective binding of live Raji cells (crystal violet stained)but not other cell types on plates coated with streptavidin horse radishperoxidase (SHRP) and biotinylated SHAL (panels E-H). (panels A-D) TheSHAL was preincubated with SAHRP and detected by DAB reagent. Lym-1binding was detected with a biotinylated anti-mouse MAb, followed bySAHRP and DAB. Panel A) SHAL on large cell lymphoma Panel B) SHAL onsmall cell lymphoma Panel C) Lym-1 MAb on large cell lymphoma and PanelD) Lym-1 MAb on small cell lymphoma. (Panels E-H) Images show selectivebinding of SHAL JP459B to live Raji cells (Panel E), but not tonon-lymphoma LnCAP (Panel F), 22RV (Panel G) or DU145 (Panel H) celllines. FIG. 17B shows that SHAL JP459B binds only to live-cultured tumorcells containing HLA-DR10, the plates are coated with streptavidin overnight, they are washed and then the SHAL is added and incubated for 2hrs and then the plates are washed again to remove unbound SHAL. Thenthe cells are added. The cells were washed and stained by Cresyl Violet.

FIG. 18 shows a tissue section from NH lymphoma illustrating that JP459Bstaining of lymphoma cells is localized to the cell membrane.

FIG. 19 illustrates ¹¹¹In-DOTA [SHAL 070804(LeacPLD)₂LPDo]biodistribution in Raji tumored mice.

FIG. 20 illustrates the binding of univalent bidentate SHAL and bivalentbidentate SHAL 070804LeacPLDB to Raji cells in presence and absence ofLym-1 antibody.

FIG. 21 illustrates various classes of SHAL targets.

FIG. 22 illustrates SHALs attached to DOTA. The red part is one ligand(deoxycholate), the green is the other ligand (5-leu-enkephalin), theblue is a lysine used to make the shortest linker, and the black is acombination of things: the PEG molecules used to make the linker betweenthe two ligands longer, a DOTA ring attached to the SHAL for binding theradioactive metal.

FIG. 23 shows formulas for JP7001.2 and triiodothyronine-deoxycholateSHALs.

FIGS. 24A and 24B show chemical structures in 2 dimensions and acronymsfor the tridentate SHAL (FIG. 24A) DvLPLLCtPCbPLDo; (dabsyl-L-valine PEGlysine lysine3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionicacid PEG 4-[4-(4-chlorobenzyl)piperazino]-3-nitrobenzenecarboxylic acidlysine-DOTA having the Ct ligand (FIG. 24A insert); and the dimeric,tridentate SHAL (FIG. 24B) (DvLPLLCtPCbPPP)₂LLDo; ((dabsyl-L-valine PEGlysine lysine3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionicacid PEG 4-[4-(4-chlorobenzyl)piperazino]-3-nitrobenzenecarboxylic acidPEG PEG PEG)₂ lysine lysine-DOTA. In various embodiments, analogbiotinylated SHALs differ only with respect to substitution of biotinfor the DOTA chelate, upper right of each SHAL.

FIGS. 25A and 25B illustrate structures of the dimeric SHAL(DvLPBaPPP)₂LLDo (FIG. 25A) and the hexa-arginine analog(DvLPBaPPP)₂LArg₆AcLLDo (FIG. 25B).

FIG. 26 illustrates binding of ¹¹¹In radiolabeled SHAL (DvLPBaPPP)₂LLDoand its hexa-arginine analog (DvLPBaPPP)₂LArg₆AcLLDo to Raji cells.Total ¹¹¹In-(DvLPBaPPP)₂LArg₆AcLLDo bound to Raji cells (unwashed),solid squares; total ¹¹¹In-(DvLPBaPPP)₂LLDo bound to Raji cells(unwashed), open squares. Cell pellets containing 10⁶ cells wereresuspended in 150 μl 5% BSA/PBS buffer containing 0-25 ng of ¹¹¹Inlabeled SHAL and incubated at RT for 1 hour. The samples werecentrifuged to separate the cells from the supernatant and both werecounted in a calibrated gamma well counter to quantify bound and unboundSHAL. Error bars are included for each data point, but in the majorityof the cases the error is smaller than the data point and the error baris not visible.

FIG. 27 shows fluorescent 3-D confocal microscopy of parent SHAL(DvLPBaPPP)₂LLDo (top row) binding to live Raji cells compared to thehexa-arginine analog (DvLPBaPPP)₂LArg₆AcLLDo (middle row). Two mid-cellfocal planes within the Raji cells are shown (left to right). Jurkat'scells treated with (DvLPBaPPP)₂LArg₆AcLLDo (left panel, bottom row) showminimal SHAL uptake. Lym-1 (right panel, bottom row) exhibits primarilycell surface membrane binding to Raji cells. The parent SHAL showsintracellular binding, while the hexa-arginine analog demonstrates notonly marked cytoplasmic binding but also intranuclear targeting. DAPI isused as the nuclear stain and AlexaFlor 610 demonstrates the location ofSHAL in these merged sequential laser images.

FIG. 28. Structure of the tridentate SHAL containing the Ct ligand shownas a 2-D schematic (upper) and as a 3-D space-filling molecularstructure (lower). The ligands used to produce this SHAL weredabsyl-L-valine (Dv),4-[4-(4-chlorobenzyl)piperazino]-3-nitrobenzenecarboxylic acid (Cb) and(3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionicacid) (Ct). The 1st lysine residue provided a free amine for covalentbinding of moieties such as metal binding macrocycles and biotin.Additional lysine residues were used to create branch points in thelinker and PEG monomers were used to provide the appropriate distancebetween the ligands. The three ligands were identified by docking tocavities in the β subunit of HLA-DR10, flanking the amino acid arginine70 shown critical for Lym-1 binding and cytotoxicity. Functionalmolecules can be incorporated at specific positions along the linker byinserting additional lysine residues, and attaching them to the freeepsilon amine of the primary lysine.

FIG. 29. Titration of SHAL cytotoxic activity in Raji human HLA-DR10expressing lymphoma cells. Absolute number of non-viable cells observed1, 2 or 3 days after addition of SHAL at concentrations shown. SHALthreshold and IC₅₀ concentrations were determined at 2 days to be 0.7and 2.5 nM (pm/ml media), respectively (mean±SD).

FIG. 30. Photographic evidence for cure in a mouse with a Rajixenograft; before SHAL treatment (upper), one week after the initial anda second 100 ng dose of SHAL i.p. By three weeks after the initial dosethe xenograft had completely regressed and represented a cure thatpersisted over the period of observation of the mouse (>84 days; lower).

FIG. 31. Overall survival effect of SHAL in mice with established Rajior Jurkat's lymphoma xenografts. Mice with Raji or Jurkat's xenograftswere treated with 100 ng SHAL i.p. once weekly for each of 3 consecutiveweeks, or untreated. Mice with treated Raji xenografts (dark circle) hadthe longest survival while mice with treated Jurkat's xenografts(square) did not survive past 56 days. Two untreated mice (empty circle)with enlarging xenografts survived 84 days.

FIG. 32. Electron micrographs of Raji xenografts excised from anuntreated mouse and a mouse 24 h after SHAL treatment. Cellularstructures were normal for this aggressive xenograft, untreated (left),whereas cells in the SHAL-treated xenograft showed condensed andfragmented chromatin and loss of cytoplasmic structures consistent withautophagic death (right) (bar, 5 μm).

DETAILED DESCRIPTION

This invention pertains to the development of a new class of bindingmolecules that can be used to specifically bind just about any targetmolecule(s). This class of binding molecules are referred to herein asSelective High Affinity Ligands or “SHALs”. The SHALs can be used in amanner analogous to antibodies in a wide variety of contexts thatinclude, but are not limited to capture reagents in affinity columns forpurification of biological or other materials, binding agents inbiosensors, agents for the assembly of nanoparticles or nanomachines,diagnostics, and therapeutics.

SHALs can also be used to detect molecular signatures that candistinguish between various pathogen types or strains. SHALs also haveuse in biodefense applications for the detection of unique proteinsignatures present in toxins and on the surfaces of pathogenic organismsand to distinguish these biothreat agents from naturally occurringnon-hazardous materials. Because the SHALs can be relatively stable whenexposed to the environment, they are particular well suited for use inbiosensors for biodefense, diagnostic, and other applications.

In certain preferred embodiments, the SHALs are used in the diagnosisand/or treatment of cancers. In such embodiments, the SHALs are directedto unique and/or specific sites (e.g., cancer-specific markers) on thesurfaces of cancers (e.g., various malignant cells).

In certain embodiments, the SHALs can have a therapeutic effect whenadministered per se (e.g., in a manner analogous to the antibodytherapeutic Herceptin™). A SHAL, like an antibody, can have a directeffect on a malignant cell that leads to cell death because the SHALserves as an agonist against a normal pathway, thereby initiating orblocking critical cell functions and leading to malignant cell death. ASHAL can also act as a vaccine because it provides malignant cellidentification either because it represents an aberrant cell surfacemarker or enhances a usual malignant cell marker.

In addition, or alternatively, the SHAL(s) can be used as targets (whenbound to the targeted cell), or as carriers (targeting moieties) forother effectors that include, but are not limited to agents such ascytotoxic agents, markers for identification by the immune system,detectable labels (for imaging), and the like.

Radioisotopes are attractive examples of cytotoxic effectors that can beattached to the carrier SHAL to selectively deliver radiotherapy to themalignant cell(s). This therapy can be administered as single agenttherapy, or in combination with marrow reconstitution in order toachieve greater dose intensity, or other drugs that may enhance theradiation effects on the malignant disease. Although there are manydifferent drugs, chemotherapeutic, biological and otherwise, that can becombined with the SHAL, taxanes are one attractive example.

Examples of cytotoxic agents include radioisotopes, immunotoxins,chemotherapeutics, enzyme inhibitors, biologicals, etc. Interestingexamples include apoptotic signals and enzymes such as the caspases.Radioisotopes represent interesting cytotoxic agents that have beenshown to be effective in conjunction with antibody antigen andligand-receptor systems. For treatment purposes, according to thepresent invention, it is considered that, in some embodiments, labelingwith a particle emitter such as beta−, beta+(positron) are preferable.In some cases, labeling with an alpha emitter or Auger-electron emitteris appropriate. There are many examples of therapeutic radioisotopesincluding yttrium-90 or iodine-131 that are of considerable currentinterest.

For certain imaging purposes, according to the present invention, it isconsidered that technetium-99, indium-111, iodine-123, or iodine-131 areattractive for single photon imaging and that beta+(positron) emitterssuch as copper-64, yttrium-86, gallium-68, etc. are particularly likelyto be very attractive when attached to a SHAL for diagnostic purposes

A SHAL consists of two or more ligands (also referred to as bindingmoieties) linked together directly or through a linker to generate acore “polydentate” molecule (SHAL) that has been designed tospecifically bind to essentially any desired target (e.g., unique orspecific sites (pockets) on an intended target malignant cell surfacemolecule). The ligands (binding moieties) comprising the SHAL caninclude essentially any moiety capable of binding a site on the target.Such binding moieties can include, but are not limited to variouschemicals (e.g., small organic molecules), proteins, sugars,carbohydrates, lectins, lipids, metals, nucleic acids, peptide andnucleic acid analogues, and the like.

Although not required, the individual ligands comprising the SHAL oftenhave relatively low affinity (e.g., less than about 10⁻⁶M) for thetarget. In contrast, the polydentate SHAL (comprising a plurality ofligands) typically shows relatively high avidity (e.g., greater thanabout 10⁻⁶M, preferably greater than about 10⁻⁸ or 10⁻⁹ or 10⁻¹⁰ M,still greater than about 10⁻¹¹ M, and most preferably greater than about10⁻¹²M).

In certain embodiments, where the target to which the SHAL is to bedirected is a protein, the ligands comprising the SHAL can be selectedto bind certain non-functional sites on the protein. A protein often hasa number (few to >50) of “pockets” or cavities distributed across itssurface. These cavities are produced as the protein chain is folded intoa three dimensional structure to make the protein functional. Thisobservation makes it possible to consider designing SHALs that exhibitmuch greater binding specificity for a given protein than previouslypossible. By linking together two moieties that bind to unique pocketson the surface of a protein with only micromolar affinities, it ispossible to design polydentate molecules (SHALs) that bind withnanomolar to picomolar affinities and are highly selective and do notcross react with other functionally related molecules. For proteins witha known or predicted structure, computational methods can be used togenerate a three-dimensional map of the molecular surface and identifysuitable sized pockets that are structurally unique for that protein asdescribed herein.

Databases containing the structures of known small molecules can bescreened for their ability to bind into pockets on the target proteinusing a “docking” program. The top candidates can then be tested using avariety of experimental techniques as described herein to identify themolecules that actually bind to the protein as well as those that bindto the correct site. Pairs or triplets of the ligands (e.g., one fromeach set) can then be attached to opposite ends of an appropriate lengthlinker using solid or solution phase chemistry to generate bidentateSHALs (FIG. 1) or tridentate SHALs. The highest affinity and mostselective SHALs can then be identified by conducting conventionalbinding studies.

It was a surprising discovery that linking together two or more, smallligands that bind weakly to target (e.g., a protein) and exhibit littleor insufficient selectivity can result in the production of a moleculethat binds to its intended target (e.g., target protein) three to six ormore orders of magnitude more tightly and with high selectivity.

Without being bound to a particular theory, it is believed that whilethe presence of two or more ligands in the SHAL would be expected toincrease the odds that the molecule might bind to a wider variety ofproteins, we have observed that this non-specific binding is weak(approximately the same as the free ligand) and those molecules attachedto non-target proteins via only one of the ligands will not remain boundlong. The enhanced affinity and selectivity observed when both ligandsin a bidentate SHAL or all the ligands comprising a polydentate SHALbind to their respective targets (e.g., pockets on a target protein) isderived from three factors that relate to the nature of the SHAL-targetinteraction: First, the presence of the linker prevents the individualligands comprising the SHAL that dissociate from their target fromdiffusing away from the target surface, increasing significantly therate at which the free ligand rebinds. Second, the reduced off rate ofrelease of the bidentate or polydentate SHAL is dictated by the factthat the probability that both (or all) ligands comprising the SHAL willsimultaneously release from their target is substantially lower than theprobability that either one will release/ The spacing between theligands comprising the SHAL which is determined by the attachmentchemistry (e.g., the linker), allows the ligands comprising the SHAL tobind simultaneously to their target only if the ligands are separated bythe correct distance. If either ligand in the SHAL binds independentlyto another target, its low affinity (1-10 micromolar affinity is typicalfor the ligands we identify) would result in the ligand falling offrapidly (the off-rate would be high). Thus the only situation in whichthe bidentate or polydentate SHAL would bind tightly to the intendedtarget (nanomolar affinity or higher) would be when both ligandscomprising the SHAL bind simultaneously to the target molecule (e.g.,target protein). Once both or all ligands are bound, the off-rate of theentire molecule (the SHAL) would be reduced dramatically. If the ligandbinding sites are selected properly (e.g., by targeting regions thatvary in amino acid sequence or structure in the case of a proteintarget), it becomes highly improbable that identical sites will be foundon another target separated by the same distance. If this extremelyunlikely event were to occur, an additional ligand binding site (e.g., athird binding site adjacent to Site 1 and -2) can be identified, and anadditional ligand can be incorporated into the SHAL (e.g., to create atridentate, quadridentate, etc. SHAL).

SHALs have certain advantages over antibodies, particularly intherapeutic and/or diagnostic applications. Typically SHALs areconsiderably smaller than antibodies. They are consequently able toachieve greater tumor penetration. In certain embodiments, they are alsoable to cross the blood brain barrier, e.g., for the treatment of braintumors. It is believed that the SHALS are also often less immunogenicthan antibodies, and are often cleared from the circulation lessrapidly.

In certain embodiments SHALs described herein are typically polydentate,i.e., the SHAL comprises two or more ligands, that are joined togetherdirectly or through one or more linkers. In certain embodiments theligands bind to different parts of the target (e.g., different epitopeson a single protein) to which the SHAL is directed. In certainembodiments the ligands bind to different molecules, e.g., differentcancer markers on a cancer cell, different proteins comprising areceptor, and the like. In certain embodiments polyspecific SHALS can beused for crosslinking the same or different antigens on the same cellthereby enhancing the signal transduction, or for pretargeting, e.g.,where one SHAL is designed to target malignant cells and is attached toother SHALs designed to “catch” a subsequently administered carrier of acytotoxic agent (e.g, chelated radiometal, etc), to recruit animmunologically active cell (e.g., macrophage, T-cell, etc.) to thesite, to activate a prodrug on the targeted SHAL, and so forth.

In certain embodiments this “multiple specificity” is achieved by theuse of polyvalent SHALs. Polyvalent SHALs are molecules in which two ormore SHALs (e.g., two or more bidentate SHALs) are joined together. Thedifferent SHALs comprising the polyvalent SHAL can be directed to thesame or different targets, e.g., as described above.

I. Construction of SHALs.

SHALs of this invention are created by identifying ligands (bindingmoieties) that bind, and in some embodiments, that specifically (orpreferentially) bind, different regions of the target molecule ormolecules. Ligands binding different regions of the target molecules arethen joined directly or through a linker to produce a bidentate SHALcomprising two different binding moieties or a polydentate SHALcomprising two or more different biding moieties. The SHAL can then bescreened for its ability to bind the intended target.

The initial identification of ligands that bind different regions of thetarget can be accomplished using virtual in silico methods (e.g.,computational methods) and/or empirical methods, e.g., as describedherein.

Once two or more suitable ligands (binding moieties) are identified theycan, optionally be screened (validated) for the ability to bind thetarget at different sites. Suitable binding ligands can then be coupledtogether directly or through a linker to form a bidentate or polydentateSHAL that can then, optionally, be screened for the ability to bind thetarget.

A) Target Selection.

Virtually any molecule, receptor, combination of molecules can serve asa target for a SHAL (see, e.g., FIG. 21). Target selection is determinedby the application for which the SHAL is intended. Thus, for example,where the SHAL is to be incorporated into an affinity column (e.g., topurify a protein or nucleic acid) the target is the molecule (e.g.,protein, nucleic acid, etc.) that is to be purified using the affinitycolumn comprising the SHAL.

Where the SHAL is to be used in the treatment and/or diagnosis of acancer, the target is typically a molecule, collection of molecules,receptor, enzyme, or other structure that is characteristic of thecancer (e.g., that permits the SHAL to preferentially bind to the cancercell as compared to a normal healthy cell).

A number of cancer-specific markers are known to those of skill in theart. Such markers include, but are not limited to Lym-1 epitope, Muc-1,C-myc, p53, Ki67, erbB-2, Her2, Her4, BRCA1, BRCA2, Lewis Y, CA 15-3,G250, HLA-DR cell surface antigen, CD2, CD3, CD7, CD19, CD20, CD22,integrin, EGFr, AR, PSA, carcinoembryonic antigen (CEA), the L6 cellsurface antigen (see, e.g., Tuscano et al. (2003) Neoplasia, 3641-3647;Howell et al. (1995) Int Biol Markers 10:126-135; Marken et al. (1992)Proc. Natl. Acad. Sci. U.S.A. 89: 3503-3507, 1992), growth factorreceptors, and/or various intracellular targets (e.g., receptors,nucleic acids, phosphokinases, etc.) and the like.

In certain embodiments, SHALs can be generated for cell surface membranetarget proteins that influence intracellular functions, therebypromoting these functions (agonist) or inhibiting these functions(antagonist) by blocking other molecular binding or causing aninhibitory or enhanced intracellular signal, e.g, phosphokinasesignaling. SHALs can be generated for cell surface membrane targetproteins such as antigens and antibodies that can be internalized intothe cell. In common with antibodies that target internalizing antigensand peptide ligands that target internalizing receptors, these SHALswill be internalized and in the cell where they can have agonist orantagonist effects on critical cell functions such as protooncogenes,phosphokinases, lysosomes and DNA/RNA/mRNA because of their agonist orantagonist functions or because they deliver a toxin or radioisotopepayload. There are several advantages of SHALs over antibodies andpeptide ligands. They include small size and the range of charge thatcan be used to permit free movement into and within the cell when anintracellular molecule is the primary target.

SHALS can be used to preferentially select specific cells by theirmembrane targets and, upon dissociation from the targeted cell surfacemembrane may freely move across the cell surface membrane to access theinside of the cell. The SHAL can be made multi-specific so that when itis internalized, or when it dissociates and penetrates the cell surfacemembrane, the second specificity can permit targeting of internal cellmolecules such as phosphokinases, lysosomal enzymes, hormone receptors,gene and proto oncogene protein products, DNA/RNA/mRNA, and the like. Incertain embodiments, uni-specific but multivalent SHALs can be generatedthat both target call surface molecules and cross-link these moleculesleading to enhanced biologic effects that have been described forcross-linked antibody-antigen systems.

In contrast to antibodies and peptide ligands that typically cannotdirectly and readily penetrate cell surface membranes, because of thesmall size of SHALs, and the ability to select the hydrophobic orhydrophilic character of the SHAL, SHALs can be produced that arecapable of penetrating the cell membrane and various intracellularcompartments. This makes it possible to generate SHALs specifically forthe purpose of targeting intracellular molecules of importance to cellfunction, such as proto oncogenes, phosphokinases, lysosomal and otherenzymes, DNA/RNA/mRNA, etc. This capability makes it possible to targetentire classes of intracellular molecules of critical importance to cellfunction, a capability not previously achievable by specific targetingmolecules. In addition to direct effects of these SHALs, they can beused as carriers of payloads, as described herein.

Proto oncogene products provide an example of a class of intracellulartargets that can be targeted by SHALs and create a useful effect incancer treatment. It is noted that Ras proteins, encoded by protooncogenes, have been targeted in vitro by antibodies injected into thecells and this blockage has resulted in cells that no longer divide.Mutation have been related to impaired control activity of theseproducts.

Many signaling pathways are susceptible to interference by the one(directly intra cellular) or two step (membrane targeting followed byinternalization) SHAL targeting of intracellular molecules. Theseinclude, but are not limited to such key pathways as “G” proteinsignaling and tyrosine specific protein kinase activity e.g., EGFR, Neu,etc. Multiple hormone receptor interventions can also be targeted bySHALs to create a change in cell function and/or in cell growth. Hormoneor enzyme targets can be bound and the function blocked. Hormonereceptor blocks that can be useful include the use of SHALs to block thebinding of estrogen like molecules to the ER (estrogen receptor) andsimilar effects to AR (androgen receptor). This would in turn interferewith DNA binding of the complex, with the resulting interference inhormone sensitive tumor cell growth and viability.

This invention also contemplates the use of SHALS to treat infectiousdiseases (e.g., AIDs, influenza, etc.) by either primary binding of theinfectious agent and/or by blocking cell invasion, and/or by blockingmetabolic pathways critical to propagation of the infectious agent(e.g., by blocking CCR5 to prevent HIV infection of cells).

Where the target to which the SHAL is to be directed comprises aprotein, in certain embodiments, at least one of the ligands (bindingmoieties) comprising the SHAL bind to a pocket in the protein. Incertain embodiments, where at least two of the binding moietiescomprising the SHAL bind to pockets of the protein and those two ligandsbind to different pockets. In certain embodiments, all of the ligandscomprising the SHAL bind to pockets in the target protein. This is notto suggest that all ligands comprising the SHAL must bind to proteinpockets. Certain embodiments are contemplated wherein one ligand bindsto a pocket and another ligand binds to a region that is not a pocket orwhere none of the ligands bind to a pocket.

When structural information is not available for a particular target(e.g., a cancer marker or a domain or epitope within a cancer marker)then the target or domain within the target can be modeled to identifyligand binding sites for the design of a SHAL for this specific target.If the target has been structurally characterized, then the crystal orNMR structure of related molecules can be used in this manner. In theevent that information is not available, a less usual circumstance, thenempirical approaches can be used to identify suitable ligands for theconstruction of a SHAL as described herein.

The empirical approaches described herein can also be used to acceleratethe process of SHAL development. IN this circumstance, modeling andother analytical steps can be performed after empirical development of aSHAL as desired for better understanding the SHAL and/or to guidesubsequent generations of SHAL development.

Although the examples illustrate one preferred embodiment where the SHALis intended as a carrier for an imaging and therapeutic radioisotopes,such as indium-111 and yttrium-90, for diagnosis and treatment, and theintended target is the HLA-DR cell surface molecule found on malignant,and normal, B lymphocytes, and therefore useful in most lymphomas andleukemias, it should be emphasized that this is only one of manyembodiments even in B cell lymphoma and leukemias. For example,antibodies are known to identify a number of other cell surface antigensknown to be attractive for antibody targeting of B cell lymphoma andleukemias. These could also be used for the same purposes and in thesame manner as those for HLA-DR. Of additional interest, the regiontargeted by SHALs designed for HLA-DR targeting, is in the area of thepit known for peptide presentation of importance to immuneidentification and rejection. Of even greater importance, antigens andepitopes have been characterized and shown to be important for antibodytargeting of adenocarcinomas of all types, including, breast, prostate,and colon malignant diseases. The aberrant mucins of the adenocarcinomasand the CEA found abundantly on many adenocarcinomas also provideattractive targets that have been rather well characterized.

In certain embodiments, it is believed the core tandem repeat protein ofmucin (MUC1) (see, e.g., FIG. 4) provides a good target forcancer-directed SHALs. MUC-1 has been shown to be upregulated andreadily available on epithelial cancers, such as prostate, breast, colonand ovarian cancers (see, e.g., FIGS. 5, 6, and 7).

Radioimmunotherapy (RIT), using Y-90 on a variety of monoclonalantibodies (mAbs), including those against MUC-1, has shown promise inpreclinical studies and trials in patients. These studies have shownthat epithelial cancers can be targeted effectively using radiolabeledmAbs, and that MUC-1 is one of the more attractive targets. Wholeantibodies, however, are not readily concentrated in and typically donot penetrate through solid cancers. Consequently the therapeutic indexfor such moieties has proven to be relatively low.

However, MUC1-directed SHALs made, e.g., according to the methodsdescribed herein, directed against the protein core tandem repeat ofMUC-1, an epitope shown in preclinical studies and RIT trials inpatients to be a unique antigenic epitope upregulated on epithelialcancers are expected to be highly effective. SHALs (selective highaffinity ligands) are quite small (˜2000 Daltons) relative to wholeantibodies (˜150,000 D), or even single chain variable fragments(˜25,000 D), so that SHALs readily penetrate and concentrate inmalignant tumors, or are rapidly cleared or excreted by the kidneys.

In certain embodiments, additional ligand(s) beyond one or two chosenfor docking sites in the epitopic region of interest are chosen fordocking sites outside the region of interest on the target. Thisprovides greater selectivity and “effective” affinity. In the case of amultimeric target, for example, HLA-DR, additional ligands can bedirected to the same or to a different subunit of the target.Specifically, in the case of HLA-DR, one or two ligands can be directedto known docking sites in the epitopic region defined for Lym-1monoclonal antibody reactivity and additional ligands can be chosen fordocking sites in the same or a different multimer, for example, the betasubunit or the alpha subunit of HLA-DR.

Other examples are SHALs with ligands chosen to react with the tandemrepeats of mucins, such MUC-1 as described above. In this instance, thecore protein is repetitive at ten mer intervals so that SHALs of similaror identical nature can be joined to provide multivalent linkage toidentical or similar but different repeats of known (or unknown)distance. Alternatively, the SHAL can have a third ligand that isidentical to one of the initial ligands but linked at a distance todocking sites at a remote region of the core tandem repeat of MUC-1.

Also, docking sites can be protrusions in addition to cavities, althoughthe latter are likely to confer greater (affinity) by virtue of thepotential for more contact interactions.

B) Compounds (Putative Ligands/Binding Moieties) to be Screened.

Virtually any agent can be screened for its ability to bind a target andthereby for its suitability for incorporation into a SHAL according tothe methods of this invention. Such agents include, but are not limitedto nucleic acids, proteins/peptides, nucleic acid or peptide analogs,metals, sugars, polysaccharides, glycoproteins, lipids, lectins, largeand small organic molecules, antibody CDRs, and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical library containing a large number ofpotential ligands (binding moieties). Such “combinatorial chemicallibraries” are then screened in one or more assays, as described hereinto identify those library members (particular chemical species orsubclasses) that display the desired binding activity. The compoundsthus identified can serve as a component of a SHAL.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biological synthesisby combining a number of chemical “building blocks” such as reagents.For example, a linear combinatorial chemical library such as apolypeptide (e.g., mutein) library is formed by combining a set ofchemical building blocks called amino acids in every possible way for agiven compound length (i.e., the number of amino acids in a polypeptidecompound). Millions of chemical compounds can be synthesized throughsuch combinatorial mixing of chemical building blocks. For example, onecommentator has observed that the systematic, combinatorial mixing of100 interchangeable chemical building blocks results in the theoreticalsynthesis of 100 million tetrameric compounds or 10 billion pentamericcompounds (Gallop et al. (1994) J. Med. Chem., 37(9): 1233-1250).

Preparation of combinatorial chemical libraries is well known to thoseof skill in the art. Such combinatorial chemical libraries include, butare not limited to, peptide libraries (see, e.g., U.S. Pat. No.5,010,175; Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493; Houghtonet al. (1991) Nature, 354: 84-88). Peptide synthesis is by no means theonly approach envisioned and intended for use with the presentinvention. Other chemistries for generating chemical diversity librariescan also be used. Such chemistries include, but are not limited to:peptoids (PCT Publication No WO 91/19735), encoded peptides (PCTPublication WO 93/20242), random bio-oligomers (PCT Publication WO92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers suchas hydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993)Proc. Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides(Hagihara et al. (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidalpeptidomimetics with a beta-D-glucose scaffolding (Hirschmann et al.,(1992) J. Amer. Chem. Soc. 114: 9217-9218), analogous organic synthesesof small compound libraries (Chen et al. (1994) J. Amer. Chem. Soc. 116:2661), oligocarbamates (Cho, et al., (1993) Science 261:1303), and/orpeptidyl phosphonates (Campbell et al., (1994) J. Org. Chem. 59: 658).See, generally, Gordon et al., (1994) J. Med. Chem. 37:1385, nucleicacid libraries (see, e.g., Strategene, Corp.), peptide nucleic acidlibraries (see, e.g., U.S. Pat. No. 5,539,083) antibody libraries (see,e.g., Vaughn et al. (1996) Nature Biotechnology, 14(3): 309-314), andPCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al. (1996)Science, 274: 1520-1522, and U.S. Pat. No. 5,593,853), and small organicmolecule libraries (see, e.g., benzodiazepines, Baum (1993) C&EN,January 18, page 33, isoprenoids U.S. Pat. No. 5,569,588,thiazolidinones and metathiazanones U.S. Pat. No. 5,549,974,pyrrolidines U.S. Pat. Nos. 5,525,735 and 5,519,134, morpholinocompounds U.S. Pat. No. 5,506,337, benzodiazepines U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.).

In certain embodiments, the initial screen for ligands (bindingmoieties) with which to build the SHAL can be performed as a virtual insilico screening method and, in this case, it is not necessary to havethe physical compounds in hand. In such instances chemical structuredatabases provide a wide range of moieties that can be screened fortheir suitability for inclusion in a SHAL as described herein.

Chemical structure databases are well known to those of skill in theart. For example, the MDL® Available Chemicals Directory (MDL ACD) ispresently the largest structure-searchable database of commerciallyavailable chemicals in the world and is available from MDL InformationSystems, Inc., San Leandro, Calif. This database is merely illustrativeand not intended to be limiting. Other chemical structure databases(e.g. ChemSpider, ZINC, etc.) are well known to those of skill in theart and include, but are not limited to various organic molecule,peptide, carbohydrate or nucleic acid structural databases.

C) Computational Identification of Ligands that Bind the Target.

Using a virtual in silico approach, computational methods can be used tocharacterize (e.g., model) the target (e.g., target protein) and toidentify molecules (binding moieties) that are expected to specificallybind to certain regions of the target. The use of computational methodsto identify molecules that specifically bind to a particular target isoften referred to as “DOCKING”.

Docking methods are well known to those of skill in the art. Twoapproaches to docking are “rigid molecule docking” in which themolecules involved are treated as rigid objects that cannot change theirspatial shape during the docking process, and soft (flexible) dockingwhere the molecules are (computationally) allowed to change shape asthey dock.

There are several physical and chemical forces that interact between thetwo molecules. These forces are used to define various docking scoresthat measure quality of each solution. These scores take into accountthe strength of these forces and the plausibility of the dockingsolution. The most significant forces typically considered in dockingalgorithms include electrical forces, van der Walls forces, and hydrogenbonds.

The docking problem is often formally stated as follows: Let A,B be tworigid molecules (e.g., the target and the potential ligand that is tobind the target) with their geometric representation in R3. We wouldlike to find a rigid transformation T:R3->R3 such that the contactsurface between T·A and B is maximal. The contact surface is typicallydefined as the surface where the distance between the molecules issmaller than a given threshold. Typically docking algorithms try toachieve a contact surface which is “large enough” instead of “maximal”and that we try to maximize not the size of the contact surface but ascore measuring the quality of the proposed docking solutions. These twoparameters are correlated but are not equivalent.

1. Rigid Docking.

One approach to the rigid docking algorithm was described by Kuntz etal. (1982) J. Mol. Biol., 161: 269-288. The Kuntz et al. algorithm isprimarily used in solving ligand-protein docking while trying to focuson ‘interesting’ sites on the surface of the molecules. The basic stagesof the algorithm involve first computing the molecular surface usingConnolly's method (see, e.g., Connolly (1983) J. Appl. Crystallography,16: 548-558; Connolly (1983) Science, 221: 709-713)). This produces aset of points on the “smoothed” molecular surface with their normals.Then a “sphere generator” (e.g., SPHGEN) is used to create a newrepresentation of the molecular surface of the target (e.g., protein)and the ligand using “pseudo-atoms” and then uses this representation tofind plausible docking sites on the molecular surface—these dockingsites that SPHGEN is looking for are cavities in the surface of thereceptor.

SPHGEN typically consists of the following five stages: First, for eachpair on Connolly points p_(i),p_(j) a sphere passing through this pairis placed such that its center is on one of the points normal. Thealgorithm then defines S_(norm)(i)={Spheres whose center is on thenormal of p_(i)}. Assuming that there n Connolly points, then for each1<=i<=n and p_(i) is on the surface of the target, we throw away all thespheres in S_(norm)(i) and leave only the one with the smallest radius.This throws all the spheres that penetrate the surface of the target.The algorithm then typically only leaves the spheres where Theta (e.g.,the angle between the pi normal and the radius from p_(j) to the centerof the sphere)<90°. Otherwise the points that define the sphere, pi andpj, are too close to each other and therefore are not located in acavity on the target's surface. Then the algorithm typically for eachatom leaves only the sphere with the maximal radius. This step leavesonly the spheres that ‘touch’ the surface of the atom. Finally, if thepoints that define the sphere, p_(i) and p_(j), belong to two differentatoms, and the distance between these atoms on the molecular sequence isless than 4—the sphere is discarded. This is done because the length ofa curve on an Alpha-helix is 3.6 and these sites are typically not to betreated as possible docking sites.

The remaining spheres are called pseudo-atoms. The next stage looks forclusters of intersecting pseudo-atoms. The existence of this kind ofcluster indicates the existence of a cavity in the molecular surface,which is considered to be a good docking site.

After all this is performed on the target the same is done on theligand, but this time we take the points and the vectors opposite totheir normals, in order to create the spheres inside the surface insteadof outside the surface. The result of SPHGEN on the target is sometimescalled the ‘negative image’ and on the ligand it's called the ‘positiveimage’. In certain embodiments, the vectors with respect to the ligandand the target can be reversed (e.g., to find elements of the targetthat dock within cavities in the ligand).

“Matching” is then typically performed. In this operation, for eachdocking site, the algorithm tries to find a transformation T that givesa good correspondence between the centers of the pseudo-atoms of thetarget to those of the ligand (in some cases, the centers of the realatoms of the ligand are used, instead of the centers of itspseudo-atoms). In some versions of DOCK, clusters of pseudo-atoms areseparated into sub-clusters in order to improve the complexity of thisstage. One way of doing this is to discard the largest sphere in thecluster, which sometimes causes the cluster to be divided into twosub-clusters

In the matching problem of rigid docking a search is performed to find atranslation and rotation of one molecule, such that good matchingbetween the interesting points in both molecules is formed. In certainembodiments, the distances between the point used instead of the pointslocations: For each molecule, the target (T) and the ligand (L), anappropriate distance matrix is defined—d^(T) _(i,j) and d^(L) _(i,j),respectively. A search is then performed to try to find two subsets in Tand L such that their distances are the same, with some tolerance oferror. These two subsets define two subgraphs with almost similardistances between their vertices. This can be done using a methodsimilar to the interpretation tree by Grimson and Lozano-Perez.

Another way of solving the matching problem is to find a “large enough”clique in a matching graph. If there are n points in the target and mpoints in the ligand, the matching graph has n*m vertices where eachvertex represents a point from the target and a point from the ligand.Let G=(V,E) be the matching graph and let u,v be vertices in V where urepresents u_(L) and u_(T) (points in the ligand and the target,respectively) and v represents v_(L) and v_(T). An edge e=(u,v) will beadded to E only when ABS[d^(L)(u_(L),v_(L))−d^(T)(u_(T),v_(T))]<tolerance. Therefore, a cliquein the matching graph defines subsets of points in the ligand and thereceptor with similar distances.

In order to evaluate the quality of the match a score is calculated. Thescore preferably takes into account the size of the contact surfacebetween the molecules and typically does not allow one molecule topenetrate the other. The DOCK algorithm uses a cubic grid that fills thebinding site and every cell in this grid has a score according to itsdistance from the centers of the receptor's atoms: 1 if the distance is2.8 Å-4.5 Å, −127 if the distance is less than 2.8 Å, and 0 if thedistance is more than 4.5 Å. (In some cases the distance 2.8 Å isreplaced by 2.4 Å). For each proposed transformation, the position ofthe ligand's points (i.e. the centers of its atoms or pseudo-atoms) inthe grid are calculated and the score is calculated as the sum of scoresof these points. Additional or alternative scores can be used variousversions of the algorithm. For example, in one version a van der Waalsenergy score can be calculated for the transformations that have goodmatching scores.

Construction of a computer model of HLA-DR 10. Using the crystalstructures that have been determined for four closely related humanHLA-DR molecules (HLA-DR 1-4), the identification of unique “pockets” onsurface of the protein, the identification of ligands that bind certainunique pockets and the construction of a SHAL using these ligands isillustrated herein in the examples.

The foregoing description is intended to be illustrative of one approachto rigid docking and is not intended to be limiting. Other approachesare known to those of skill in the art. It is noted that the SPHGEN andDOCK programs are commercially available (e.g., directly from theUniversity of California and various commercial manufacturers ofsoftware).

2. Soft (Flexible) Docking.

Soft docking algorithms are also well known to those of skill in the art(see, e.g., Jiang and Kim (1991) J. Mol. Biol., 219: 79-102;Katchalski-Katzir et al. (1992) Proc. Natl. Acad. Sci. U.S.A., 89(6):2195-2199, etc.).

In the method described by Jiang and Kim, supra, an enumeration on the6-dimensional space of rigid transformation is performed and thesetransformations are given scores according to their energetic value.Both molecules are placed on a grid and the matching is evaluated usingthe distances between grid cells, the number of penetrations and thedirections of the points' normals. The algorithm works on the output ofConnolly's algorithm and works on the entire molecular surface (i.e. nocavities are looked for—as opposed to the DOCK algorithm). In order todecrease the enumeration, the algorithm typically uses tworesolutions—low and fine. The low resolution uses ˜0.3 points per squareangstrom, and the fine resolution uses ˜1 point per square angstrom.

Each cell in the grid is marked as “surface” (if it contains at leastone Connolly point) or “volume” (if it doesn't contain any Connollypoint). Usually, each surface cell contains 2-3 Connolly points.

An enumeration on the rotations of one of the molecules (usually smallerone) is performed. For each rotation the following is performed: Thesurface and volume cell of the molecule is calculated. Assuming thatthere's at least one pair of surface cells (one from each molecule) thatare matched by the transformation, an enumeration on all of these pairsis performed. For each pair the transformation is calculated and it isevaluated by checking the directions of the normals, the number ofsurface-to-surface matches and the number of penetrations. The goodtransformations are those who have a small number of penetrations and alot of surface-to-surface matches. This is done first in low resolutionand the best results are calculated again in fine resolution with theaddition of an approximated energetic score. The approximated energeticscore is calculated according to the number of “favorable” and“unfavorable” interactions. There are several categories for the atomsof each molecule and combinations of these categories are marked as“favorable” if they have a good contribution to the energeticplausibility of the match, or “unfavorable” otherwise.

For example, it is unfavorable that an atom with positive charge isplaced near another atom with positive charge, but it is favorable iftwo atoms are adjacent if one of them is an H-donor and the other is anH-acceptor.

The approach of Katchalski-Katzir et al., supra, is to enumerate on thepossible translations, while using FFT to calculate the matching scoreefficiently. Similar to the previous algorithm, both molecules areplaced on a 3-dimensional grid, but here 3 types of grid cells aredefined—“volume”, “surface” and “intermediate”. If the molecules are Aand B, the matrices A_(l,m,n) and B_(l,m,n) are defined as follows(l,m,n are the grid coordinates): A_(l,m,n)={1—if (l,m,n) is a “surface”cell, q—if (l,m,n) is an “intermediate” cell, 0—otherwise}, andB_(l,m,n)={1—if (l,m,n) is a “surface” cell, r—if (l,m,n) is an“intermediate” cell, 0—otherwise}.

In certain embodiments, parameters are chosen such that q<0 and r>0while |q| is large and |r| is small. The scalar product of thesematrices can be efficiently calculated using FFT thus improving thealgorithm's performance considerably.

Again, it is noted that the foregoing description is intended to beillustrative of one approach to rigid docking and is not intended to belimiting. Other approaches are known to those of skill in the art. Forexample, additional approaches/programs include, but are not limited to:FlexX from Tripos (http://www.biosolveit.de/FlexX/) which is commonlyused for high-throughput screening. It uses an empirical scoringfunction. It allows for flexible docking by rotating around torsionalbonds. It is sold as a module of the Sybyl program (distributed byTripos, Inc., St. Louis). GOLD from CCDC(http://www.ccdc.cam.ac.uk/prods/gold/) which uses a genetic algorithmto generate conformers for a ligand. It also enables customization ofthe torsional energy within smaller fragments of the molecule and canaccommodate local protein flexibility. Autodock UCSD(http://www.scripps.edu/pub/olson-web/doc/autodock/) uses a Lamarckiangenetic algorithm to generate conformers for a ligand. AutoDock is bestused when there are only a few ligands and the binding energies need tobe more accurate. Some good reviews on Docking include Lyne (2002) DrugDiscovery Today. 7 (20): 1047, and Taylor et al. (2002) J.Computer-Aided Mol. Design., 16: 151.

D) Empirical Approaches and Verification of Ligand Binding.

The use of computational methods to identify ligands for use in theconstruction of a SHAL requires at least some information regarding thestructure of the target molecule(s). This invention also contemplatesthe use of methods that require no knowledge regarding the structure ofthe target to which the SHAL is to be directed.

In certain “empirical” embodiments, individual ligands or libraries ofligands are screened against the target molecule(s) and/or cells,bacteria, viruses, etc. displaying the target molecule(s) to identifyligands that bind the desired target (at least low affinity). Ligandsare identified that bind to different regions of the target molecule(s).In certain embodiments, ligands are identified that bind to differentregions of the target molecules and that do not exclude each other fromsuch binding.

Ligands that can simultaneously bind to the target without excludingeach other can then be joined together, directly or through a linker, tocreate a polydentate SHAL which can, optionally, be subsequentlyscreened for the ability to bind to the target molecule(s), e.g., athigh affinity.

In addition to use in empirical approaches for ligand identification,physical screening methods are also desirable for validating binding ofligands identified using the virtual in silico approaches discussedabove. In addition, it can be desirable to additionally determine thebinding orientation of two or more ligands, e.g., to confirm that theligands bind to different sites on the target and/or to estimate spacingwhen the ligands are incorporated into a SHAL.

Assays for detecting the binding of one or more ligands to a target arewell known to those of skill in the art. For example, in one simpleembodiment, the ligands can be labeled with a detectable label andcontacted with the target molecule(s) which are immobilized on asubstrate. After a wash, detection of the labels in association with theimmobilized target molecule(s) indicates that the ligands bind to thetarget. In certain embodiments, different ligands can be labeled withdifferent labels (e.g., different color fluorescent labels), and thesimultaneous binding of multiple ligands can be visualized.

Alternatively, competitive binding assays can be performed. In suchassays the target molecule(s) are contacted with one ligand known tobind the target. The target is also contacted with the “test” ligand andthe ability of the test ligand to bind to the target in the presence ofthe first ligand is evaluated.

Fluid phase assays can also be performed. For example, the ligand(s) andthe targets can be labeled with different labels. The ligands can becontacted to the target molecule(s) and binding of the two can readilybe evaluated, e.g., using a flow cytometer. Flow cytometry methods arewell known to those of skill in the art (see, e.g., Omerod (1994) FlowCytometry: A Practical Approach. IRL Press, Oxford; Shapiro PracticalFlow Cytometry. 3rd Edition. Alan R Liss, Inc.; Givan (1992) FlowCytometry. First Principles. Wiley-Liss, New York; Robinson (1993)Handbook of Flow Cytometry Methods, Wiley-Liss, New York, and the like).

Determination of ligand binding and orientation can also be determinedusing a number of different methods. These include, but are not limitedto Saturation Transfer Difference nuclear magnetic resonance (Mayer andMeyer (1999) Angew Chem Int Edit, 38:1784-1788) and Transfer NOE (trNOE)nuclear magnetic resonance (NMR) spectroscopy (Henrichsen et al. (1999)Angew Chem Int Edit., 38:98-102; Cosman et al. (2002) Chem Res Toxicol15: 1218-1228). These methods can be used to screen the ligands inmixtures of several to several hundred per experiment to determine whichligands bind to the target molecule(s) e.g., under biologically relevantconditions and to determine which ligands bind to the same (ordifferent) sites. Diffusion experiments (Lin et al. (1997) J. OrganicChem., 62: 8930-8931) can also be performed with those ligands that havebeen determined to bind in order to assess the relative binding affinityof each compound.

Other approaches to detecting binding of the ligands to the targetmolecule(s) include, but are not limited to surface plasmon resonance(BIAcore assay), saturation transfer difference nuclear magneticresonance spectroscopy, other nuclear magnetic resonance spectroscopymeasurements, mass spectrometry, capture microarrays, bead-based libraryassays, and other physical binding assays.

The foregoing assays are intended to be illustrative and not limiting.Using the teaching provided herein numerous other assays for detectingligand binding to the target molecule(s) will be known to those of skillin the art.

Following the identification of a set of ligands that bind to the targetmolecule(s) (e.g., HLA-DR10), competition experiments can be performed,e.g., by NMR to determine if they bind to one of the pockets comprisingthe target molecule(s) (e.g., in the case of HLA-DR10, to one of thepockets encompassing the Lym-1 epitope.

As indicated above, this can readily be accomplished by preparing acomplex between the target and a known binding ligand and determining ifa second ligand can bind the complex. Thus, for example, the case ofHLA-DR10 target, a Lym-1:HLA-DR10 complex can be prepared and the set ofligands that bind to HLA-DR10 can be re-tested to determine if they willstill bind to the protein when the Lym-1 antibody is bound.

Those ligands that no longer bind to the Lym-1:HLA-DR10 complex can beidentified (these ligands bind to the unique sites that distinguishHLA-DR10 from the other HLA-DR molecules) and used in a second set ofcompetition experiments to identify those molecules that bind todifferent sites within the Lym-1 epitope. In experiments conducted withpairs of ligands, transfer nuclear Overhauser effects (trNOE) that occurbetween the bound ligands and the HLA-DR10 protein (Cosman et al. (2002)Chem Res Toxicol 15: 1218-1228), in the absence of the Lym-1 antibody,can be used to identify those ligands that bind to the same anddifferent sites. Bound ligands exhibit negative NOE signals, whileunbound ligands have positive signals (Id.). If both ligands in the pairare observed to be bound to the protein at the same time, the resultswill indicate that the two ligands must bind to different sites. Thescreening thus can readily identify sets of ligands that bind todifferent sites within the target molecule(s) (e.g., to different sites(Site 1 and Site 2) within the Lym-1 epitope of HLA-DR10).

After sets of ligands have been identified that to bind to differentsites on the target, the orientation of the ligands in the binding sitescan, optionally, be further evaluated using classical molecular dynamicssimulations. The methods of molecular dynamics simulations are clearlydescribed in the Examples. For example, for each ligand, one to threeorientations within the binding pocket can be simulated for 500 psec.This will help determine which functional groups on the ligands arelikely to be in contact with the target and which functional groups areaccessible by solvent. This information can be used to identify analogswith modified or different functional groups that can be tested fortheir ability to bind to the target and confirm that a particularfunctional group can be used as the site for linker attachment withoutdisrupting the binding of the ligand to the target.

The use of these approaches to identify ligands that bind to specificsites on various targets is described in the literature. For example,these methods have been used to identify ligands that bind to specificsites on the targeting domain of tetanus neurotoxin (Cosman et al.(2002) Chem Res Toxicol 15: 1218-1228; Lightstone et al. (2000) Chem.Res. Toxicol., 13: 356-362) as well as eleven ligands we've alreadyidentified that bind to HLA-DR10.

Applicants' previous studies using a similar approach to identifyligands that bind to two sites on the targeting domain of tetanusneurotoxin (Id.) have required screening less than 30 ligandsexperimentally. Over half of the ligands predicted to bind to theprotein were observed to bind experimentally. Thus we believe thatscreening a set of 30 ligands per site should provide a sufficientnumber of compounds that bind to initiate SHAL synthesis. However, if insome embodiments, a suitable number of ligands (e.g., 3-5) are notidentified to bind in the first round of NMR screens, additional sets ofligands can be selected and screened until suitable ligands are foundthat bind to two different sites in the target.

In certain embodiments, the selection of the ligand pairs (or othermulti-ligand combinations) to be linked together is based on thefollowing criteria (in descending order of importance): 1) binding site(the two ligands that comprise a pair preferably bind to differentsites); 2) reliability of information obtained on available functionalgroups that can be used to attach the molecules to the linker withoutdisrupting binding (analogs with known derivatives that are confirmed tobind, indicating the modification of particular functional group doesnot affect binding, are given priority); 3) the ligand's expected easeof attachment to the linker (the ligands preferably have functionalgroups that facilitate asymmetric attachment chemistry to put adifferent ligand on opposite ends of the linker); 4) relative bindingaffinity, e.g., as determined from the NMR diffusion experiments(ligands exhibiting the strongest binding to the protein are typicallyselected first); 5) known information on their toxicity in animals orhumans (priority is typically given to use of ligands that are known tobe non-toxic or have already been approved for use in otherpharmaceuticals); and 6) ligand cost.

Because it is likely that a number of the individual ligands (bindingmoieties) may bind weakly to other proteins, it will not be necessary(or meaningful) to prescreen the individual ligands to determine if theybind to other proteins that might be encountered in the circulationbefore they are used to create SHALs. The binding of any single ligand(half of the pair used to create a bidentate SHAL) to other proteins isexpected to be weak (micromolar at best). Consequently, the off-rate ofthe molecule will be high and the SHAL that only binds through oneligand to other proteins will be quickly removed from the tissue andcirculation. High affinities (and low off-rates) typically will only beobtained when both ligands in the pair bind simultaneously to sitesseparated by the proper distance, which is dictated by the length of thelinker connecting them. These two criteria will only be met when theSHAL comes in contact with the intended target. Once bound, the off-rateis expected to be reduced 1000 to 1,000,000 fold (based on previousstudies) over that observed for either ligand alone. For this reason,cross-reactivity is not expected to be a significant complication.

E) Combined Computational/Empirical Approaches.

The binding of a SHAL to the region(s) of its target is based upon fitand charge and is dependent on the 3D structure and constituents of thebinding moieties. In the computational approach described above,generating SHALs can involve definition/identification of attractiveregion(s) on the target molecule. Thus, for example, proteins of alltypes, including antigens, receptors and signaling proteins, can bemodeled to find docking sites and ligands. The ligands can subsequentlybe tested using empiric methods. These methods typically requireknowledge of the constituent molecules to be included in the modeling.

The empirical methods described above, rely on screening of libraries(e.g., combinatorial libraries) of potential ligands to find suitablebinders. In this approach, no foreknowledge is required beyondavailability of a target (e.g., protein, cell, etc.) of interest.Libraries are experimentally culled for binders. This can be followed bycompetition with a molecule, such as an antibody, peptide or chemical(ligand) known to react with the molecule in the region chosen to betargeted. This approach permits the elimination of binding chemicals orpeptides that are not of interest and definition of those that bind tothe region of interest.

A third approach is intermediate in nature and uses foreknowledge ofattractive target molecules and regions of these molecules for initialcompetitive screening and counter-screening. Thus, for example, initialtargets or ligand-library constituents can be computationallypredicated. The optimized target or target collection and/or optimizedlibrary can then be screened and counter-screened as described herein toidentify optimal binders.

F) Bead-Based Library Screening.

One approach for screening for ligands that bind the target moleculesinvolves producing a combinatorial library comprising a large number ofpotential ligands each attached to a different bead/solid support. Thecombinatorial library can be a “random” library, or can be synthesizedto provide numbers of variant having, e.g., particular (e.g., optimized)core chemistry.

Combinatorial synthetic methods are well known and are used to rapidlymake large “libraries” of distinct compounds. In various embodiments thestarting material (e.g., an amino acid) is covalently anchored to solidsupport. This is followed by the stepwise addition of monomers(typically protected monomers) such as amino acids, nucleotides, smallorganic molecules, and the like. Millions of distinct molecules can becreated by varying number of steps and number of reactants (e.g., in asplit-mix synthesis approach), but typically each bead contains only onecompound.

The compounds comprising the library can be screened while still boundto the beads. Colorimetric, fluorometric, radiographic methods or othermethods can be used to visualize positive (binding) beads. These can becaptured (e.g., with a pipette, with a metal bar if the beads aremagnetic), and the compound can then be characterized.

Thus, for example, one can synthesize a library of peptide ligands thatbind HLA-DR10 molecules. Peptide synthesis chemistry is well developed.However, to obtain peptide ligands that have a longer half-life in vivo,one might choose to produce a peptide ligand library where the peptidescomprise D-amino acids. Such peptides are expected to be more resistantto proteolysis in vivo. Moreover, D-amino acids are generally considerednon-toxic.

The Lym-1 epitope on HLA-DR10 is highly polar. Thus, in synthesizing thelibrary of potential binders on can select polar D-amino acids forsynthesis (e.g., Ser, Asp, etc.) Using Split/Mix synthesis (see, e.g.,U.S. Pat. No. 5,574,656) a library of D-peptides bound to beads iscreated.

Then HRP-tagged HLA-DR10 is added to the bead mixture. The HRP colorlabel is visualized and the positive beads are removed. The positivebeads can then be tested against, e.g., HLA-DR10 positive cell lines.Beads that test positive in this assay can then be tested against, e.g.,a tissue panel to ensure that binding is HLA-DR10 specific. The specificbinders in this assay can then be characterized (e.g., sequenced usingEdman degradation, mass spectrometry, etc.).

Alternatively, there are strategies for encoding the identity of eachthe compound during the synthesis of the library (see, e.g., U.S. Pat.Nos. 5,565,324; 5,723,598; 5,834,195; 6,060,596; 6,503,759; 6,507,945;6,721,665; 6,714,875; and the like). Using such “tagging” strategies theidentity of the positive binders can then readily be determined.

G) Linking the Ligands (Binding Moieties) to Produce a Polydentate SHAL.

Once two more ligands (binding moieties) are identified that bind todifferent sites on the target, the ligands are linked either directly orthrough a linker to produce a polydentate SHAL. Where only two ligandsare joined the SHAL is bidentate. Where three ligands are joined theSHAL is tridentate, and so forth.

A number of chemistries for linking molecules directly or through alinker are well known to those of skill in the art. The specificchemistry employed for attaching the ligands (binding moieties) to eachother to form a SHAL will depend on the chemical nature of the ligand(s)and the “interligand” spacing desired. Ligands typically contain avariety of functional groups e.g., carboxylic acid (COOH), free amine(—NH2) groups, that are available for reaction with a suitablefunctional group on a linker or on the other ligand to bind the ligandthereto.

Alternatively, the ligand(s) can be derivatized to expose or attachadditional reactive functional groups. The derivatization may involveattachment of any of a number of linker molecules such as thoseavailable from Pierce Chemical Company, Rockford Ill.

A “linker”, as used herein, is a molecule that is used to join two ormore ligands (binding moieties) to form a polydentate SHAL. The linkeris typically chosen to be capable of forming covalent bonds to all ofthe ligands comprising the SHAL. Suitable linkers are well known tothose of skill in the art and include, but are not limited to, straightor branched-chain carbon linkers, heterocyclic carbon linkers, aminoacids, nucleic acids, dendrimers, synthetic polymers, peptide linkers,peptide and nucleic acid analogs, carbohydrates, polyethylene glycol andthe like. Where one or more of the ligands comprising the SHAL arepolypeptides, the linkers can be joined to the constituent amino acidsthrough their side groups (e.g., through a disulfide linkage tocysteine) or through the alpha carbon amino or carboxyl groups of theterminal amino acids.

In certain embodiments, a bifunctional linker having one functionalgroup reactive with a group on a the first ligand and another groupreactive with a functional group on a second ligand can be used to formthe desired SHAL. Alternatively, derivatization may involve chemicaltreatment of the ligand(s), e.g., glycol cleavage of the sugar moiety ofglycoprotein, carbohydrate or nucleic acid with periodate to generatefree aldehyde groups. The free aldehyde groups can be reacted with freeamine or hydrazine groups on a linker to bind the linker to the ligand(see, e.g., U.S. Pat. No. 4,671,958). Procedures for generation of freesulfhydryl groups on polypeptide, such as antibodies or antibodyfragments, are also known (See U.S. Pat. No. 4,659,839).

In certain embodiments, lysine, glutamic acid, and polyethylene glycol(PEG) based linkers different length are used to couple the ligands. Anumber of SHALs have been synthesized using a combination of lysine andPEG to create the linkers (see, e.g., Examples and FIG. 13). Chemistryof the conjugation of molecules to PEG is well known to those of skillin the art (see, e.g., Veronese (2001) Biomaterials, 22: 405-417;Zalipsky and Menon-Rudolph (1997) Pp. 318-341 In: Poly(ethyleneglycol)Chemistry and Biological Applications. J. M. Harris and X. Zalipsky(eds), Am. Chem. Soc. Washington, D.C.; Delgado et al. (1992) DrugCarrier Syst., 9: 249-304; Pedley et al. (1994) Br. J. Cancer, 70:1126-113-0; Eyre and Farver (1991) Pp. 377-390 In: Textbook of ClinicalOncology, Holleb et al. (eds), Am. Cancer Soc., Atlanta Ga.; Lee et al.(1999) Bioconjug. Chem., 10: 973-981; Nucci et al. (1991) Adv. DrugDeliv., 6: 133-151; Francis et al. (1996) J. Drug Targeting, 3:321-340).

One advantageous feature of the synthetic scheme used to create theseSHALs is that the approach allows the attachment of almost any type ofmolecule to a third site on the linker. In the first round of SHALsynthesis, biotin has been attached at this site to facilitate the invitro binding studies. The biotin tag makes it possible to quicklymeasure the binding to the isolated protein by surface plasmon resonanceand examine the selectivity of the SHAL for binding to live cells andtissue sections.

Once the SHAL has been tested and confirmed to bind to the target (e.g.,HLA-DR10), metal chelators such as DOTA (or other effectors) can beattached in the final round of synthesis to enable the delivery ofradionuclides or other effectors to target bearing cells (e.g., tumorcells).

After retesting the effector-SHAL conjugates to reconfirm their abilityto bind to the target, the conjugates exhibiting the best selectivityfor their targets can, optionally be tested for their biodistribution intest organisms (e.g., mice). Other unique molecules can also be attachedto this site in future studies so these same SHALs can also be used, forexample, to test the utility of pre-targeting approaches forradioisotope delivery.

H) Stepwise Solid-Phase SHAL Synthesis.

In certain embodiments, SHAL synthesis proceeds by a stepwise-solidphase synthesis approach. In this approach each linker component orligand is attached onto a growing molecule (SHAL) covalently attached tothe surface of a resin. After each chemical reaction the resin can beextensively washed to remove the unreacted products.

In one approach, DOTA was attached to the linker at the beginning of thesynthesis. After the excess DOTA was washed away, multiple additionalchemical reactions that were carried out on the resin to add the variouslinkers and ligands, and after each reaction the unreacted products wereagain washed away. By the time the synthesis of the SHAL was completed,the amount of free DOTA present in the sample was undetectable whenexamined by HPLC and mass spectroscopy. The DOTA link is extremelystable, so it does not come off the SHAL once it's been attached.

I) Screening SHALs for Affinity and Selectivity.

In certain embodiments, a library of SHALS comprising different ligands(binding moieties) and/or comprising different length linkers isscreened to identify those SHALS that have the best affinity and/orselectivity for the target. Such screening assays can be performed in anumber of formats including, but not limited to screening for binding toisolated targets, screening for binding to cells in culture, screeningfor binding to cells in tissue arrays, and screening for in vivo bindingto the desired target.

1. SHAL Binding to Isolated Targets (e.g., Proteins).

In certain embodiments, the binding affinities of the best SHALs can beestimated by mass spectrometry of the SHAL-target complexes, followed bya more accurate surface plasmon resonance (SPR) spectroscopy (Shuck(1997) Annu Rev Biophys Biomol Struct., 26: 541-566; Van Regenmortal(2001) Cell Mol Life Sci., 58: 794-800) measurement of the SHAL-targetbinding affinity using for example, the IASYS Plus or BiaCoreinstruments. In order to perform the SPR measurement, biotin can beadded to the linker through a third functional group (as describedabove) and the SHAL can be bound to commercially available streptavidincoated chips. In certain preferred embodiments, only those SHALsexhibiting nM or higher binding affinities can be considered useful. TheSHALs exhibiting the greatest affinity can then be tested for theirselectivity. Experiments can be performed to test the selectivity ofSHAL binding to targets in the presence of molecules related to thetargets. Thus, for example, where the SHAL is directed to HLA-DR10, theSHAL can be evaluated for its ability to bind target molecules in thepresence of Raji cell surface proteins extracted and separated byaffinity chromatography. After treating the gel with the biotinylatedSHAL and rinsing out excess unbound SHAL, the location of the bound SHALcan be detected by staining with Rhodamine tagged streptavidin. Incertain embodiments, the SHALs that are considered to exhibit reasonableprotein selectivity can be those molecules in which 95% or more of thefluorescence is associated with the HLA-DR10 monomer and multimer peaks.

2. SHAL Binding to Cells in Culture.

Where the SHAL target is a marker on a cell (e.g., a cancer cell marker)it may be desired to assess the specificity of binding of the SHAL tointact cells.

Cell binding studies can be conducted with the biotinylated (orotherwise labeled) SHALs, using for example the fluorescence of boundRhodamine-tagged streptavidin to confirm the SHALs bind to target (e.g.,Raji) cells. If the SHAL is observed to bind, SPR measurements can beconducted to determine the affinity of intact cells to the SHAL. Incertain embodiments, those SHALs that exhibit at least a 2-fold,preferably at least a 5-fold, and more preferably at least a 10-folddifference in the staining intensity of target (e.g., tumor) cells overcontrols can be selected for further testing and development. Analogs ofthe most promising SHALs can be synthesized with a DOTA moleculeattached to the linker, and binding experiments can be conducted usingradionuclide-tagged SHALs to obtain more quantitative data and alsoattempt to determine if the SHAL is retained on the surface of the cellor is internalized using NanoSIMS or other methods. This information isuseful in making decisions about the type of radioisotope that is to beloaded into the chelator. If the SHAL remains on the surface, the SHALis typically utilized alone or with effectors that do not requireinternalization (e.g., alpha emitters such as ⁹⁰Yttrium, variousdetectable labels, and the like). If evidence is obtained to suggest theSHAL is internalized upon binding to the target cells, it is possible toutilize the SHAL with effectors that are active when internalized.

3. Analysis of Cell Selectivity Using Tissue Arrays.

Tissue array technology can be used to screen SHALs to determine tissuespecificity (e.g., malignant and normal tissue reactivity in the case ofanti-tumor SHALs). Tissue arrays are well known to those of skill in theart (see, e.g., Kononen et al. (1998) Nat Med., 4:844-847; Torhorst etal. (2001) Am J Pathol., 159: 2249-2256; Nocito et al. (2001) Int JCancer, 94: 1-5, and the like). In its basic form, a tissue microarraysis formed by taking small cores of each individual tumor case/block andassembling these cores into a single block (Id.). By sectioning this newblock, standard immunohistochemistry and in-situ hybridizationtechniques can be used. Therefore, one can assay hundreds of tissuesamples in one experiment rather than having to perform hundreds ofdifferent experiments. FIGS. 2 and 3 outline how the tissue arrays canbe used and show a diagram of illustrative tissue array.

In one embodiment, for the normal tissue array we have identified 80unique tissues, which include oropharynx, heart, lung, stomach, spleen,liver, kidney, intestine, bone marrow, pancreas, bladder, muscle,adrenal, breast, brain, normal prostate and skin for placement on thetissue microarray. For a lymphocyte specific tissue array, we haveincluded neoplastic lymphocytic lines, xenografts, and patient materialcollected from the Human Biological Specimen Repository at UC Davis.Using these or similar tissue arrays, one can determine the non-specificbinding of the SHALs to normal tissue and specific binding tolymphocytic and prostatic neoplasms. The hybridization of the SHALs tothe tissue arrays is straightforward. Using biotinylated SHALS asdescribed herein, labeled streptavidin (e.g., Rhodamine-taggedstreptavidin) can readily be used to identify those cells that bind theSHALs. When required, the tissue microarray results can be verified byconventional histology and immunohistology.

In one illustrative approach, 2-4 tissue cylinders, with a diameter of0.6-mm, can be punched from the selected areas of each “donor” tissueblock and brought into a recipient paraffin block in order to assemblethe tissue microarray, using a Tissue Microarrayer (e.g., BeecherInstruments, Silver Spring, Md.) and the techniques described by Kononenet al. (1998) Nat Med., 4:844-847. Tissue microarray slides containing,for example, 200-400 cores can then be sectioned at a thickness of 4 μm.Routine Hematoxylin & Eosin staining can be performed in order to verifythat each core represents its selected histopathology. Forimmunohistochemistry, microwave in a citrate buffer can be used forantigen retrieval. The images of the slides can be captured by confocalscanner (ScanScope, Mountain View, Calif.) and visualized with MrSidViewer 2.0 (LizardTech, Inc.) as described below. The ability to viewthe tissue array images on a computer rather than a microscopedramatically increases the efficiency of analysis.

The major obstacle to digital pathology has been the representation ofglass slides in a digital format. Unlike radiology, which begins with adigital representation of a patient rendered by CT, MRI, or now “digitalplain film”, pathology requires that all tissue samples be processed andmade into stained tissue sections mounted on glass slides forinterpretation. The new technology produces images of the entire glassslide, thereby producing a true digital representation of the entirehistopathologic specimen (Whole Slide Imaging). Most of the currentinstruments use a microscope equipped with a digital camera and arobotic stage to capture thousands of individual images. Each image isfocused by the content expert. Once acquired, these images are typicallybe stitched (or tiled) together to form the final representation of theslide. This process is both very time consuming and, due to the highnumber of images involved, the images are often misaligned.

ScanScope, (Aperio® Technology) is a new type of digital slide scannerthat scans a microscope slide in 3 to 5 minutes, capturing 8 to 12gigabyte images at 50,000 dpi. The images are then compressed, processedand stored for presentation (see below). MrSid® by Lizardtech®compresses the large, 8 to 12 gigabyte images using a proprietarymulti-layer wavelet Jpeg format with compression ratios reaching 20:1without significant image degradation. The images can then be eitherviewed locally or served from a web server. Unlike standard web-basedstill images, which are typically downloaded to be viewed within abrowser, the MrSid processed images are viewed from the web, and thebrowser application never downloads the entire image. Because the imagesare acquired at their maximal resolution, “lower” magnification views ofan image are constructed by the server. Using the combination ofScanScope and Zoomify browser, entire slides (12 gigabytes) can becaptured and processed in less than 20 minutes.

4. In Vivo Analysis of Selectivity.

Of course in vivo selectivity of a SHAL can also readily be determined.This can be accomplished by administering the SHAL to a test animal(e.g., a laboratory rat) comprising a cell or tissue that displays thetarget to which the SHAL is directed. After sufficient time, the animalcan be sacrificed and the target tissue(s) and normal tissues examined(e.g., histologically) to evaluate the specificity and amount of SHALdelivery. In certain embodiments, the SHAL can be coupled to an imagingreagent that permits non-invasive imaging and thereby permit theevaluation of real time pharmacodynamics.

By way of illustration, pharmacokinetic and radiation dosimetric mousestudies can be performed, e.g., on the SHALs illustrated in theExamples, to generate data upon which to select one for clinical trialsof pharmacokinetics and radiation dosimetry in patients, usingestablished methods. Pharmacokinetics can be performed in female nudemice bearing Raji human lymphoma xenografts of defined size usingestablished methods (DeNardo et al. (1998) Clin. Cancer Res., 4:2483-2490; Kukis et al. (1995) Cancer Res., 55: 878-884). Mice can beinjected with DOTA-tagged SHALs containing ¹¹¹In or ⁹⁰Y and mice can besacrificed, e.g., at each of at least 5 time points to provide samplesfor analysis. Initial studies can be conducted at the extremes of earlyand late time points expected for these small molecules so that theintermediate time points can be determined. Data for peptides can beused to define the extreme time points. When using ¹¹¹In or ⁹⁰Y as atracer, the longest time point would typically be about 5 days. Totalbody clearances can be determined using a sodium iodide detector system.Blood clearance can be monitored by taking periodic blood samples fromthe tail veins of the mice. At the time of sacrifice, the xenograft andnormal tissues can be removed, weighed and counted in a gamma wellcounter to provide organ distribution data.

In order to assess SHAL dose (mass) effect, studies can be conducted at,e.g., 5 dose levels, once again beginning with small and large SHALamounts to guide selection of the intermediate amounts to be studied.Because of the novelty of the SHALs, selection of study time points anddose levels will typically be guided by information available forantibody (e.g., Lym-1) studies in mice and for, e.g., somatostatinreceptor peptide ligands.

In certain embodiments, the ideal pharmacokinetics and dosimetry toachieve with our SHALs are those that approach what has beenaccomplished using sodium iodide (NaI) in the treatment of thyroidtumors. The SHALs should be small enough to completely penetrate themalignancy and be readily excreted in the urine. Typically at least anorder of magnitude better target recognition and binding affinity tolymphomas and leukemias than current antibodies will provide the desiredtumor cell selectivity. While the rapid clearance of smaller molecules,such as the SHALs, from the circulation might be considered adisadvantage, the remarkable effectiveness of NaI in treating thyroidtumors has shown this “disadvantage” can be turned into an advantage ifthe reagent has the right combination of affinity and selectivity. Ifthe SHALs are taken up well, target only a specific family of cells(e.g., B lymphocytes and their malignant relatives), bind tightly withlow off-rates, and are cleared rapidly from the system, the dosereceived by normal tissue (relative to malignant) should besubstantially lower than that obtained using existing targetingantibodies.

Using established methods (DeNardo et al. (2000) J. Nucl. Med., 41:952-958; DeNardo et al. (1999) J. Nucl. Med., 40: 1317-1326; DeNardo etal. (1999) J. Nucl. Med., 40: 302-310; Shen et al. (1994) J. Nucl. Med.,35: 1381-1389; Siegel (1994) J. Nucl. Med., 35: 1213-1216) asguidelines, protocols can readily be developed for conductingpharmacokinetic and radiation dosimetry studies in patients withlymphomatous diseases of the B cell type or other cancers. ProjectedSHAL dose (mass) levels can be determined using the data generated inmice and adjusted for the relative BSA of mice and patients using knownmethods (see, e.g., Freireich et al. (1996) Cancer Chemother. Rep., 50:219-244). In certain embodiments, a protocol is selected that providesthe optimal dose level using information on the therapeutic indices fortumor to marrow for a nonmyeloablative strategy and tumor todose-limiting non-marrow organ for a myeloablative strategy.

J) Optimization of SHAL Affinity, Selectivity and Metabolism by Varyingthe Linker Length and Linker and Ligand Structure.

SHAL affinity, selectivity and metabolism can be optimized by varyingthe linker length, and/or the linker and ligand structure, usingcomputer modeling and experimental studies. Linker lengths can bereduced or increased to improve the SHAL's affinity for its target.Changes in the individual ligands used to create the SHAL or alterationsin individual ligand structure can also be made to improve binding,target selectivity and optimize the clearance of unbound SHAL from theorganism. Modifications in the structure of the linker itself can alsobe considered to facilitate SHAL clearance, if necessary, from normaltissues and peripheral blood through the incorporation of cleavablebonds (e.g., a peptide or other cleavable linker) that attach thechelator to the SHAL.

If a particular SHAL is observed to exhibit non-specific binding (e.g.,to many proteins in the cell extracts or to both Raji and controlcells), additional SHALs can be synthesized using different pairs ofligands until a suitably specific SHAL is identified.

1. Maximization of SHAL Binding Affinity for Target Molecule(s).

Binding affinity of multidentate reagents to protein or cell surfacetargets can be increased by one to several orders of magnitude bychanging and optimizing the length of the linker separating the ligands.Without being bound to a particular theory, it is believed that thisincrease is related to achieving the optimal separation between theligands to allow them to bind to their individual sites as well as toproviding sufficient rotational flexibility within the linker itself toenable the optimal interaction of each ligand within its binding site(e.g., binding pocket).

In certain embodiments, the initial linker length that is chosen for usein the initial SHALs is identified by estimating the distance betweenthe two (or more) bound ligands that are to be linked together. Once ithas been determined that a particular combination of linked ligandsactually binds to the target, additional modeling can be conducted tofurther refine the length of the linker and optimize the SHALs bindingaffinity.

For example, where the target is HLA-DR10, the structure of the HLA-DR10beta subunit can modeled with both ligands bound in their respectivepockets and various length PEG linkers interconnecting the ligands (see,e.g., the Examples herein). From molecular dynamics studies theorientations of the bound ligands can be evaluated to improve the linkerdesign. Further molecular dynamics simulations can be performed toinclude the linkers and the ligands, thus simulating the polydentateligands interacting with the target, e.g., as described herein.

Once the results of these modeling experiments are obtained, anadditional set of SHALs can be synthesized with linkers spanning therange of sizes predicted to be optimal, and their binding affinities canbe experimentally tested.

2. Optimization of Target Selectivity and Metabolism of SHAL.

Computational methods can also be sued to determine if changes in thestructure of the individual ligands that are linked together to producethe SHAL improve target selectivity and optimize SHAL metabolism and itsclearance from normal tissues and peripheral circulation. This can beaccomplished, for example, by examining the types of functional groupspresent inside a targeted binding pocket and their location relative tothe bound ligand.

Molecular dynamics studies can be conducted using differentconformations of the ligand and selected ligand analogs to aid theidentification of ligand derivatives that fit optimally into eachbinding site (e.g., pocket). Diffusion NMR experiments (Lin et al.(1997) J. Organic Chem., 62: 8930-8931) can be conducted to compare andrank the affinities of a subset of the ligand analogs. The particularanalogs chosen for analysis are typically selected based on the resultsprovided by computer modeling and the analog's commercial availabilityor ease of synthesis. If higher affinity analogs are identifiedexperimentally, a set of new SHALs can be synthesized and tested fortheir affinity, selectivity in binding to targets, and desirablemetabolic properties (e.g., rapid clearance from peripheral circulation,liver and kidney).

In certain embodiments, the small size of the SHAL can result in itsbeing cleared from the tissues too quickly to be effective in deliveringa suitable amount of effector to the target cells. If this is observed,various approaches can be used to optimize the retention time of theSHAL in the target tissue. One involves using a biotin attachment siteon the linker to add a third ligand that binds to another site on thetarget. This is expected to increase the affinity of the SHAL tosubpicomolar levels and reduce the off-rate of the bound moleculedramatically. Alternatively, the effective size of the SHAL can beincreased substantially by attaching it to larger, multi-arm PEGmolecules and/or to other molecules.

K) Illustrative SHALs.

Using the teachings provided herein, a number of different SHALs thatbind, for example cancer markers (e.g., HLA-DR10) can readily beprepared. In various embodiments the SHALS include, but are not limitedto bidentate SHALS (comprising two binding ligands), tridentate SHALS(comprising three binding ligands), tetradentate SHALS (comprising fourbinding ligands), pentadentate SHALS (comprising five binding ligands),and so forth. In various embodiments can be multimeric (e.g., structuresand/or complexes comprising two, three, four, five, or more SHALs). TheSHALs can be homomultimeric (comprising two, three, four, five, or moreof the same type of SHAL), or heteromultimeric (comprising, for example,two, three, four, or five or more SHALS where at least two are differentspecies of SHAL).

In certain embodiments the SHALS comprise one or more, preferably two ormore, or three or more ligands described in any of Tables 1, 5, 6, 7, or8 and/or analogues thereof. Certain preferred SHALS include, but are notlimited to bidentate SHALS (see, e.g. FIGS. 22 and 23), tridentate SHALS(see, e.g., FIG. 24A), dimeric bidentate SHALS, bimeric (bis) tridentateSHALS (see, e.g., FIG. 24B), and the like. In certain embodiments, theSHALS comprise one or more of the ligands shown in Table 1.

TABLE 1 Illustrative ligands that can be included in SHALS as describedherein. Some of these (marked with *) could be used in place of the Ctligand or attached to the linker as a fourth component, functioning notto help the SHAL bind to the protein better but as an inhibitor once theSHAL gets inside the cell. 1 BOC-4-aminomethyl-L-Phe 2*4[[5-(Trifluoromethyl)pyridin-2-yl]oxy]phenyl]N-phenylcarbamate 3*(R)-2-[4-(5-chloro-3-fluoro-2-pyridyloxy)phenoxy]propionic acid 4*2-(((3-chloro-5(trifluoromethyl)pyridin-2-yloxy)phenoxy)methyl)acrylates5*2-(((3-chloro-5(trifluoromethyl)pyridin-2-yloxy)phenyl)methyl)acrylates6*2-(((3-chloro-5(trifluoromethyl)pyridin-2-yloxy)phenyl)methyl)acrylonitriles7*3-(3-chloro-4-{[5-(trifluoromethyl)-2-pyridinyl]oxy}anilino)-3-oxopropanoicacid 8 *Sethoxydim 9 *Clethodim 10 *5-(Tetradecyloxy)-2-furoic acid 11*2-[(2,6-Dichlorophenyl)amino]benzeneacetic acid 12*2-[4-(4-Chlorophenoxy)phenoxy]propanoic acid 13*(RS)-2-{4-[3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy}propanoicacid 14 *(RS)-2-[4-(6-chloro-1,3-benzoxazol-2-yloxy)phenoxy]propanoicacid 15 *(RS)-2-[4-(2,4-dichlorophenoxy)phenoxy]propanoic acid 16*(RS)-2-{4-[5-(trifluoromethyl)-2-pyridyloxy]phenoxy}propanoic acid 17*(RS)-2-[4-(6-chloroquinoxalin-2-yloxy)phenoxy]propanoic acid 18*(RS)-2-[4-(α,α,α-trifluoro-p-tolyloxy)phenoxy]propanoic acid 195-([4,6-Dichlorotriazin-2-yl]amino)fluorescein hydrochloride 203-[N-(4-acetylphenyl)carbomoyl]pyridine-2-Carboxylic acid 213-(2-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy}anilino)-3-oxopropanoicacid 22 L-ornithine-beta-alanine 232-Methyl-1-(3-morpholinopropyl)-5-phenyl-1H-pyrrole-3-carboxylic acid 24Hippuric acid 25 Hippuryl-D-lysine 26 Hippuryl-L-phenylalanine

Certain illustrative SHALS are also shown in Table 2.

TABLE 2 List of illustrative multidentate and dimeric, multidentateSHALs and their molecular weight (MW in Daltons). DOTA on the SHALcontributes an additional 400 (or 244 for biotin) Daltons. AcronymIdentity MW Multidentate LeacPLD acetylated 5-leuenkephalin PEG lysinedeoxycholate 1,505 ItPLD triiodothyronine PEG lysine deoxycholate 1,559DvLPBaPL dabsyl-L-valine lysine PEG N-benzoyl-L-arginyl-4-amino- 1,317benzoic acid PEG lysine CtLPTPL3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3- 1,163oxopropanionic acid lysine PEG L-thyronine PEG lysine CtLPBaPL3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3- 1,287oxopropanionic acid lysine PEG N-benzoyl-L-arginyl-4- amino-benzoic acidPEG lysine DvPLLCtPCbL dabsyl-L-valine PEG lysine lysine3-(2-([3-chloro-5- 1,765trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionic acid PEG4-[4-(4-chlorobenzyl)piperazino]-3- nitrobenzenecarboxylic acid lysineDimeric, Multidentate (LeacPLD)₂LP (acetylated 5-leuenkephalin PEGlysine deoxycholate)₂ lysine 3,006 PEG (ItPDP)₂LL (triiodothyronine PEGdeoxycholate PEG)₂ lysine lysine 3,113 (DvLPBaP)₂LL (dabsyl-L-valinelysine PEG N-benzoyl-L-arginyl-4- 2,626 aminobenzoic acid PEG)₂ lysinelysine (DvLPBaPP)₂LL (dabsyl-L-valine lysine PEG N-benzoyl-L-arginyl-4-2,921 aminobenzoic acid PEG PEG)₂ lysine lysine (DvLPBaPPP)₂LL(dabsyl-L-valine lysine PEG N-benzoyl-L-arginyl-4- 3,210 aminobenzoicacid PEG PEG PEG)₂ lysine lysine (DvLPBaPPPP)₂LL (dabsyl-L-valine lysinePEG N-benzoyl-L-arginyl-4- 3,501 aminobenzoic acid PEG PEG PEG PEG)₂lysine lysine (DvLCsPBaPPP)₂CsLL (dabsyl-L-valine lysine cysteic acidPEG N-benzoyl-L-arginyl- 3,666 4-aminobenzoic acid PEG PEG PEG)₂ cysteicacid lysine lysine (DvPLLCtPCbPPP)₂LL (dabsyl-L-valine PEG lysine lysine3-(2-([3-chloro-5- 4,267trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionic acid PEG4-[4-(4-chlorobenzyl)piperazino]-3- nitrobenzenecarboxylic acid PEG PEGPEG)₂ lysine lysine

It was discovered that various SHALS described herein are effectivealone to inhibit the growth and/or proliferation of a cancer cell and incertain instances kill the cancer cell. The SHALS can thus be usedalone, or attached to one or more effectors, e.g., as described herein.

L) SHALS Attached to Transduction Peptides.

In certain embodiments the SHALs include (e.g., are attached to) one ormore transduction peptides. A transduction peptide is a peptide thatacts as a “transmembrane shuttle” facilitating entry of the peptide intoa cell (e.g., facilitating penetration of the cell membrane).Transduction peptides are well known to those of skill in the art. Suchpeptides include, but are not limited to the nuclear localization signal(NLS) of simian virus 40 (SV40) T antigen ((Yoneda (1997) J. Biochem.,121: 811-817), the protein transduction domain of HIV Tat protein (Tatpeptide) (Vives et al. (1997) J. Biol. Chem. 272: 16010-16017; Schwarzeet al. (1999) Science 285: 1569-1572; Torchilin et al. (2003) Proc.Natl. Acad. Sci., USA, 100: 1972-1977.), the integrin-binding peptide(RGD peptide) (Hart et al. (1994) J. Biol. Chem. 269: 12468-12474), theheparin-binding domain of vitronectin (VN peptide) (Vogel et al. (1993)J. Cell Biol. 121: 461-468), antennapedia protein of Drosophila (see,e.g., Joliot et al. (1991) Proc. Natl. Acad. Sci., USA, 88: 1864-1868),penetratin (Tseng et al. (2002) Mol Pharmacol., 62: 864-887), intactproteins that naturally pass through cell membranes (the herpes virusprotein VP22 (Phelan et al. (1998) Nat Biotechnol., 16: 440-443),synthetic cationic peptide transporters such as oligoarginine (Tung andWeissleder (2003) Adv. Drug Delivery Rev., 55: 281-294; Futaki (2005)Adv. Drug Delivery Rev., 57: 547-558), lactosylated poly-L-lysine(Midoux et al. (1993) Nucl Acids Res., 21: 871-878), short peptidesequences selected from phage display libraries (Kamada et al. (2007)Biol Pharm Bull. 30: 218-223; see also peptides 1-6 in Table 3) thatexhibit sequence similarities to know peptide shuttles, and the like.

TABLE 3 Amino acid sequences of illustrative transduction peptides.Peptide Sequence Seq ID No. 1 S-G-E-H-T-N-G-P-S-K-T-S-V-R-W-V-W-D  9 2S-M-T-T-M-E-F-G-H-S-M-I-T-P-Y-K-I-D 10 3Q-D-G-G-T-W-H-L-V-A-Y-C-A-K-S-H-R-Y 11 4M-S-D-P-N-M-N-P-G-T-L-G-S-S-H-I-L-W 12 5S-P-G-N-Q-S-T-G-V-I-G-T-P-S-F-S-N-H 13 6S-S-G-A-N-Y-F-F-N-A-I-Y-D-F-L-S-N-F 14 8G-T-S-R-A-N-S-Y-D-N-L-L-S-E-T-L-T-Q 15 Tat13 G-R-K-K-R-R-Q-R-R-R-P-P-Q16 Antennapedia R-Q-I-K-I-WF-Q-N-R-R-M-K-WK-K 17 VP22N-A-K-T-R-R-H-E-R-R-R-K-L-A-I-E-R 18 hexa-Arg R-R-R-R-R-R 19

The foregoing list of transduction peptides is intended to beillustrative and not limiting. Other transduction peptides will be knownto and readily available to one of ordinary skill in the art and usingthe teaching provided herein can readily be incorporated into/attachedto a SHAL. A review of illustrative transduction peptides is provided byDerossi et al. (1998) Trends Cell Biol. 8: 84-87.

II. Chimeric Moieties Comprising SHALs (e.g., Cancer Specific SHALs).

The SHALS of this invention are selected to specifically bind toparticular targets. Where the targets are markers characteristic of aparticular cell type (e.g., a tumor cell) the SHALS can be used tospecifically deliver one ore more effectors to the target cell.

In certain embodiments, the SHALs specifically bind to cancer cells. Inthese embodiments, the SHALS can be used alone as therapeutics (e.g., toinhibit growth and/or proliferation of a cancer cell) and/or they can becoupled to an effector to provide efficient and specific delivery of theeffector (e.g., an effector molecule such as a cytotoxin, a radiolabel,etc.) to various cancer cells (e.g., isolated cells, metastatic cells,solid tumor cells, etc.).

In certain preferred embodiments, the SHALs of this invention areutilized in a “pretargeting” strategy (resulting in formation of achimeric moiety at the target site after administration of the effectormoiety) or in a “targeting” strategy where the SHAL is coupled to aneffector molecule prior to use to provide a chimeric molecule.

A chimeric molecule or chimeric composition or chimeric moiety refers toa molecule or composition wherein two or more molecules that existseparately in their native state are joined together to form a singlemolecule having the desired functionality of its constituent molecules.Typically, one of the constituent molecules of a chimeric molecule is a“targeting molecule” in this instance one or more SHALs. The targetingmolecule acts to direct the chimeric molecule to its particular target,e.g., a cancer cell.

Another constituent of the chimeric molecule is an “effector”. Theeffector molecule refers to a molecule or group of molecules that is tobe specifically transported to the target cell (e.g., a cancer cell). Itis noted that in this context, such specific transport need not beexclusively to or into a cancer cell, but merely need to providepreferential delivery of the effector to, or into, the cancer cell ascompared to normal healthy cells.

The effector molecule typically has a characteristic activity that is tobe delivered to the target cell. Effector molecules include, but are notlimited to cytotoxins, labels, radionuclides, ligands, antibodies,drugs, liposomes, nanoparticles, viral particles, cytokines, and thelike.

In certain embodiments, the effector is a detectable label, withpreferred detectable labels including radionuclides. Among theradionuclides and labels useful in the radionuclide-chelator- (e.g.,biotin) conjugates of the present invention, gamma-emitters,positron-emitters, x-ray emitters and fluorescence-emitters are suitablefor localization, diagnosis and/or staging, and/or therapy, while betaand alpha-emitters and electron and neutron-capturing agents, such asboron and uranium, also can be used for therapy.

The detectable labels can be used in conjunction with an externaldetector and/or an internal detector and provide a means of effectivelylocalizing and/or visualizing prostate cancer cells. Suchdetection/visualization can be useful in various contexts including, butnot limited to pre-operative and intraoperative settings. Thus, incertain embodiment this invention relates to a method ofintraoperatively detecting cancers in the body of a mammal. Thesemethods typically involve administering to the mammal a compositioncomprising, in a quantity sufficient for detection by a detector (e.g.,a gamma detecting probe), a cancer specific SHAL labeled with adetectable label (e.g., antibodies of this invention labeled with aradioisotope, e.g., ¹⁶¹Tb, ¹²³I, ¹²⁵I, and the like), and, afterallowing the active substance to be taken up by the target tissue, andpreferably after blood clearance of the label, subjecting the mammal toa radioimmunodetection technique in the relevant area of the body, e.g.,by using a gamma detecting probe.

The label-bound SHAL can be used in the technique of radioguidedsurgery, wherein relevant tissues in the body of a subject can bedetected and located intraoperatively by means of a detector, e.g., agamma detecting probe. The surgeon can, intraoperatively, use this probeto find the tissues in which uptake of the compound labeled with aradioisotope, that is, e.g., a low-energy gamma photon emitter, hastaken place.

In addition to detectable labels, preferred effectors include cytotoxins(e.g., Pseudomonas exotoxin, ricin, abrin, Diphtheria toxin, and thelike), or cytotoxic drugs or prodrugs, in which case the chimericmolecule cam act as a potent cell-killing agent specifically targetingthe cytotoxin to cancer cells.

In still other embodiments, the effector can include a liposomeencapsulating a drug (e.g., an anti-cancer drug such as doxorubicin,vinblastine, taxol, etc.), an antigen that stimulates recognition of thebound cell by components of the immune system, an antibody thatspecifically binds immune system components and directs them to thecancer, and the like.

A) Certain Preferred Effectors.

1) Imaging Compositions.

In certain embodiments, the chimeric molecules of this invention can beused to direct detectable labels to a tumor site. This can facilitatetumor detection and/or localization. In certain particularly preferredembodiments, the effector component of the chimeric molecule is a“radioopaque” label, e.g., a label that can be easily visualized usingx-rays. Radioopaque materials are well known to those of skill in theart. The most common radiopaque materials include iodide, bromide orbarium salts. Other radiopaque materials are also known and include, butare not limited to organic bismuth derivatives (see, e.g., U.S. Pat. No.5,939,045), radiopaque polyurethanes (see U.S. Pat. No. 5,346,981,organobismuth composites (see, e.g., U.S. Pat. No. 5,256,334),radiopaque barium polymer complexes (see, e.g., U.S. Pat. No.4,866,132), and the like.

The SHALs of this invention can be coupled directly to the radiopaquemoiety or they can be attached to a “package” (e.g., a chelate, aliposome, a polymer microbead, etc.) carrying or containing theradiopaque material as described below.

In addition to radioopaque labels, other labels are also suitable foruse in this invention. Detectable labels suitable for use as theeffector molecule component of the chimeric molecules of this inventioninclude any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels in the present invention include magnetic beads (e.g.,Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, texasred, rhodamine, green fluorescent protein, and the like), radiolabels(e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ¹⁸F, etc.) or other tags for imaging,enzymes (e.g., horse radish peroxidase, alkaline phosphatase and otherscommonly used in an ELISA), and colorimetric labels such as colloidalgold or colored glass or plastic (e.g., polystyrene, polypropylene,latex, etc.) beads.

Various preferred radiolabels include, but are not limited to ⁹⁹Tc,²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ^(113m)In, ⁹⁷Ru, ⁶²Cu, 64lCu, ⁵²Fe,^(52m)Mn, ⁵¹Cr, ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ¹²¹Sn, ¹²⁷Te,¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵¹Pm ¹⁵³Sm,¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh, and ¹¹¹Ag.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm, scintillation detectors, and the like. Fluorescent markers may bedetected using a photodetector to detect emitted illumination. Enzymaticlabels are typically detected by providing the enzyme with a substrateand detecting the reaction product produced by the action of the enzymeon the substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

2) Radiosensitizers.

In another embodiment, the effector can be a radiosensitizer thatenhances the cytotoxic effect of ionizing radiation (e.g., such as mightbe produced by ⁶⁰Co or an x-ray source) on a cell. Numerousradiosensitizing agents are known and include, but are not limited tobenzoporphyrin derivative compounds (see, e.g., U.S. Pat. No.5,945,439), 1,2,4-benzotriazine oxides (see, e.g., U.S. Pat. No.5,849,738), compounds containing certain diamines (see, e.g., U.S. Pat.No. 5,700,825), BCNT (see, e.g., U.S. Pat. No. 5,872,107),radiosensitizing nitrobenzoic acid amide derivatives (see, e.g., U.S.Pat. No. 4,474,814), various heterocyclic derivatives (see, e.g., U.S.Pat. No. 5,064,849), platinum complexes (see, e.g., U.S. Pat. No.4,921,963), and the like.

3) Radioisotopes.

In certain embodiments, the effector comprises one or more radioisotopesthat when delivered to a target cell bring about radiation-induced celldeath.

For medical purposes, the most important types of decay are gammaemission, beta decay, alpha decay, and electron capture. The gammaemitted by a radionuclide, such a ¹³¹I, exits the body, allowing the useof external scintigraphic imaging to determine the biodistribution ofradiolabeled antibodies (the optimal energy range for immunoscintigraphyis 100-250 keV). In contrast, beta particles deposit most of theirenergy within a few millimeters of the point of decay. Beta emissionsfrom radionuclides such as ¹³¹I or ⁹⁰Y that have targetedantigen-positive tumor cells can kill nearby antigen-negative tumorcells through a “crossfire” effect.

Yttrium-90, a pure beta emitter, has several properties that make it anattractive choice for radioimmunotherapy: 1) a high beta energy(E_(max)=2.29 MeV; maximum range of particulate energy in tissue=11.9mm) which enables it to kill adjacent tumor cells; 2) metal chemistry,which facilitates the synthesis of radioisotope-antibody conjugates anduse of a pretargeting approach; and 3) a sufficiently long physicalhalf-life (2.67 days) for use with intact SHALs, which may take 1-3 daysto reach their peak concentration in tumors.

In certain embodiments, the effector can include an alpha emitter, i.e.a radioactive isotope that emits alpha particles and/or anauter-electron emitter. Alpha-emitters and auger-electron emitters haverecently been shown to be effective in the treatment of cancer (see,e.g., Bodei et al. (2003) Cancer Biotherapy and Radiopharmaceuticals,18:861). Suitable alpha emitters include, but are not limited ²¹²Bi,²¹³Bi, ²¹¹At, and the like.

Table 4 illustrates some radionuclides suitable for radioimmunotherapy.This list is intended to be illustrative and not limiting.

TABLE 4 Illustrative radionuclides suitable for radioimmunotherapy. Max.particulate Radio- Decay Physical energy nuclide mode half life (%)Advantages Disadvantages I-131 β, γ  8 d 807 keV (1)* Iodine chemistry,Dehalogenation, 606 keV (86)* inexpensive radiation safety 336 keV(13)*concerns Cu-67 β, γ 62 h 577 keV (20)* Images, metal Scarce 484 keV(35)* chemistry, long 395 keV(45)* retention in tumor Lu-177 β, γ 6.7 d 497 keV (90)* Images Scarce, bone seeker 384 keV (3)* 175 keV(7)* Re-186β, γ, 91 h 1.07 MeV (77)* ^(99m)Tc chemistry Scarce electron 934 keV(23)* capture Y-90 β 64 h 2.29 MeV (100)* Metal chemistry Doesn't image,bone seeker Re-188 β 17 h 2.13 MeV (100)* Scarce, short ½ life Bi-212 α,β  1 h 6.09 MeV (27)** Doesn't image, short 6.05 MeV (70)** ½ life,unstable 5.77 MeV (2)** daughter product 5.61 MeV (1)** At-211 α,  7 h5.87 MeV High RBE, Doesn't image, short electron (100)** hypoxia lesss ½life, unstable capture important, short daughter product range I-125Electron 60 d 35 keV (100) Short range Doesn't image, long capture ½life *beta irradiation **alpha irradiation RBE, relative biologiceffectiveness

4) Ligands.

In various embodiments the effector molecule can also be a ligand, anepitope tag, or an antibody. Particularly preferred ligand andantibodies are those that bind to surface markers on immune cells.Chimeric molecules utilizing such antibodies as effector molecules actas bifunctional linkers establishing an association between the immunecells bearing binding partner for the ligand or antibody and theprostate cancer cell(s).

5) Chelates

Many of the pharmaceuticals and/or radiolabels described herein arepreferably provided as a chelate, particularly where a pre-targetingstrategy is utilized. The chelating molecule is typically coupled to amolecule (e.g., biotin, avidin, streptavidin, etc.) that specificallybinds an epitope tag attached to a prostate cancer specific antibody ofthis invention.

Chelating groups are well known to those of skill in the art. In certainembodiments, chelating groups are derived from ethylene diaminetetra-acetic acid (EDTA), diethylene triamine penta-acetic acid (DTPA),cyclohexyl 1,2-diamine tetra-acetic acid (CDTA),ethyleneglycol-O,O′-bis(2-aminoethyl)-N,N,N′,N′-tetra-acetic acid(EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid(HBED), triethylene tetramine hexa-acetic acid (TTHA),1,4,7,10-tetraazacyclododecane-N,N′—,N″,N′″-tetra-acetic acid (DOTA),hydroxyethyldiamine triacetic acid (HEDTA),1,4,8,11-tetra-azacyclotetradecane-N,N′,N″,N′″-tetra-acetic acid (TETA),substituted DTPA, substituted EDTA, and the like (see, e.g., FIG. 22).

Examples of certain preferred chelators include unsubstituted or,substituted 2-iminothiolanes and 2-iminothiacyclohexanes, in particular2-imino-4-mercaptomethylthiolane.

One chelating agent, 1,4,7,10-tetraazacyclododecane-N, N, N″,N′″-tetraacetic acid (DOTA), is of particular interest because of itsability to chelate a number of diagnostically and therapeuticallyimportant metals, such as radionuclides and radiolabels.

Conjugates of DOTA and proteins such as antibodies have been described.For example, U.S. Pat. No. 5,428,156 teaches a method for conjugatingDOTA to antibodies and antibody fragments. To make these conjugates, onecarboxylic acid group of DOTA is converted to an active ester which canreact with an amine or sulfhydryl group on the antibody or antibodyfragment. Lewis et al. (1994) Bioconjugate Chem. 5: 565-576, describes asimilar method wherein one carboxyl group of DOTA is converted to anactive ester, and the activated DOTA is mixed with an antibody, linkingthe antibody to DOTA via the epsilon-amino group of a lysine residue ofthe antibody, thereby converting one carboxyl group of DOTA to an amidemoiety.

Alternatively, the chelating agent can be coupled, directly or through alinker, to an epitope tag or to a moiety that binds an epitope tag.Conjugates of DOTA and biotin have been described (see, e.g., Su (1995)J. Nucl. Med., 36 (5 Suppl):154P, which discloses the linkage of DOTA tobiotin via available amino side chain biotin derivatives such asDOTA-LC-biotin or DOTA-benzyl-4-(6-amino-caproamide)-biotin). Yau etal., WO 95/15335, disclose a method of producing nitro-benzyl-DOTAcompounds that can be conjugated to biotin. The method comprises acyclization reaction via transient projection of a hydroxy group;tosylation of an amine; deprotection of the transiently protectedhydroxy group; tosylation of the deprotected hydroxy group; andintramolecular tosylate cyclization. Wu et al. (1992) Nucl. Med. Biol.,19(2): 239-244 discloses a synthesis of macroyclic chelating agents forradiolabeling proteins with ¹¹¹In and ⁹⁰Y. Wu et al. makes a labeledDOTA-biotin conjugate to study the stability and biodistribution ofconjugates with avidin, a model protein for studies. This conjugate wasmade using a biotin hydrazide which contained a free amino group toreact with an in situ generated activated DOTA derivative.

It is noted that the macrocyclic chelating agent1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) binds⁹⁰Y and ¹¹¹In with extraordinary stability. Kinetic studies in selectedbuffers to estimate radiolabeling reaction times under prospectiveradiopharmacy labeling can be performed to determine optimalradiolabeling conditions to provide high product yields consistent withFDA requirements for a radiopharmaceutical. It is also noted thatprotocols for producing Yttrium-90-DOTA chelates are described in detailby Kukis et al. (1998) J. Nucl. Med., 39(12): 2105-2110.

6) Cytotoxins.

The SHALs of this invention can be used to deliver a variety ofcytotoxic drugs including therapeutic drugs, a compound emittingradiation, molecules of plants, fungal, or bacterial origin, biologicalproteins, and mixtures thereof. The cytotoxic drugs can beintracellularly acting cytotoxic drugs, such as short-range radiationemitters, including, for example, short-range, high-energy α-emitters asdescribed above, or enzyme inhibitors.

Enzymatically active toxins and fragments. thereof are exemplified bydiphtheria toxin A fragment, nonbinding active fragments of diphtheriatoxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin Achain, modeccin A chain, .alpha.-sacrin, certain Aleurites fordiiproteins, certain Dianthin proteins, Phytolacca americana proteins (PAP,PAPII and PAP-S), Morodica charantia inhibitor, curcin, crotin,Saponaria officinalis inhibitor, gelonin, mitogillin, restrictocin,phenomycin, and enomycin, for example.

Particularly preferred cytotoxins include Pseudomonas exotoxins,Diphtheria toxins, ricin, and abrin. Pseudomonas exotoxin A (PE) is anextremely active monomeric protein (molecular weight 66 kD), secreted byPseudomonas aeruginosa, which inhibits protein synthesis in eukaryoticcells through the inactivation of elongation factor 2 (EF-2) bycatalyzing its ADP-ribosylation (catalyzing the transfer of the ADPribosyl moiety of oxidized NAD onto EF-2).

The toxin contains three structural domains that act in concert to causecytotoxicity. Domain Ia (amino acids 1-252) mediates cell binding.Domain II (amino acids 253-364) is responsible for translocation intothe cytosol and domain III (amino acids 400-613) mediates ADPribosylation of elongation factor 2, which inactivates the protein andcauses cell death. The function of domain Ib (amino acids 365-399)remains undefined, although a large part of it, amino acids 365-380, canbe deleted without loss of cytotoxicity (see, e.g., Siegall et al.(1989) J. Biol. Chem. 264: 14256-14261).

Where the SHAL is attached to PE, one preferred PE molecule is one inwhich domain Ia (amino acids 1 through 252) is deleted and amino acids365 to 380 have been deleted from domain Ib. However all of domain Iband a portion of domain II (amino acids 350 to 394) can be deleted,particularly if the deleted sequences are replaced with a linkingpeptide such as GGGGS (SEQ ID NO:20).

In addition, the PE molecules can be further modified usingsite-directed mutagenesis or other techniques known in the art, to alterthe molecule for a particular desired application. Means to alter the PEmolecule in a manner that does not substantially affect the functionaladvantages provided by the PE molecules described here can also be usedand such resulting molecules are intended to be covered herein.

For maximum cytotoxic properties of a preferred PE molecule, severalmodifications to the molecule are recommended. An appropriate carboxylterminal sequence to the recombinant molecule is preferred totranslocate the molecule into the cytosol of target cells. Amino acidsequences which have been found to be effective include, REDLK (SEQ IDNO:21) (as in native PE), REDL (SEQ ID NO:22), RDEL (SEQ ID NO:23), orKDEL (SEQ ID NO:24), repeats of those, or other sequences that functionto maintain or recycle proteins into the endoplasmic reticulum, referredto here as “endoplasmic retention sequences”. See, for example,Chaudhary et al. (1991) Proc. Natl. Acad. Sci. USA 87:308-312 andSeetharam et al, J. Biol. Chem. 266: 17376-17381. Preferred forms of PEcomprise the PE molecule designated PE38QQR. (Debinski et al. Bioconj.Chem., 5: 40 (1994)), and PE4E (see, e.g., Chaudhary et al. (1995) J.Biol. Chem., 265: 16306).

Methods of cloning genes encoding PE and coupling such cytotoxins totargeting moieties are well known to those of skill in the art (see,e.g., Siegall et al. (1989) FASEB J., 3: 2647-2652; and Chaudhary et al.(1987) Proc. Natl. Acad. Sci. USA, 84: 4538-4542, and referencestherein).

Like PE, diphtheria toxin (DT) kills cells by ADP-ribosylatingelongation factor 2 thereby inhibiting protein synthesis. Diphtheriatoxin, however, is divided into two chains, A and B, linked by adisulfide bridge. In contrast to PE, chain B of DT, which is on thecarboxyl end, is responsible for receptor binding and chain A, which ispresent on the amino end, contains the enzymatic activity (Uchida et al.(1972) Science, 175: 901-903; Uchida et al. (1973) J. Biol. Chem., 248:3838-3844).

In a preferred embodiments, the SHAL-Diphtheria toxin chimeric moleculesof this invention have the native receptor-binding domain removed bytruncation of the Diphtheria toxin B chain. Particularly preferred isDT388, a DT in which the carboxyl terminal sequence beginning at residue389 is removed. Chaudhary et al. (1991) Bioch. Biophys. Res. Comm., 180:545-551. Like the PE chimeric cytotoxins, the DT molecules can bechemically conjugated to the prostate cancer specific antibody, but, incertain preferred embodiments, the antibody will be fused to theDiphtheria toxin by recombinant means (see, e.g., Williams et al. (1990)J. Biol. Chem. 265: 11885-11889).

7) Viral Particles.

In certain embodiments, the effector comprises a viral particle. TheSHAL can be conjugated to the viral particle e.g., via a proteinexpressed on the surface of the viral particle (e.g., a filamentousphage). The viral particle can additionally include a nucleic acid thatis to be delivered to the target (prostate cancer) cell. The use ofviral particles to deliver nucleic acids to cells is described in detailin WO 99/55720.

8) Other Therapeutic Moieties.

Other suitable effector molecules include pharmacological agents orencapsulation systems containing various pharmacological agents. Thus,the SHAL can be attached directly to a drug that is to be delivereddirectly to the tumor. Such drugs are well known to those of skill inthe art and include, but are not limited to, doxorubicin, vinblastine,genistein, an antisense molecule, and the like.

Alternatively, the effector molecule can comprise an encapsulationsystem, such as a viral capsid, a liposome, or micelle that contains atherapeutic composition such as a drug, a nucleic acid (e.g., anantisense nucleic acid or another nucleic acid to be delivered to thecell), or another therapeutic moiety that is preferably shielded fromdirect exposure to the circulatory system. Means of preparing liposomesattached to antibodies are well known to those of skill in the art (see,for example, U.S. Pat. No. 4,957,735, Connor et al. (1985) Pharm. Ther.,28: 341-365) and similar methods can be used for coupling SHALs.

B) Attachment of the SHAL to the Effector.

One of skill will appreciate that the SHALs of this invention and theeffector molecule(s) can be joined together in any order. Thus, invarious embodiments, the effector can be attached to any ligandcomprising the SHAL and/or to the linker joining the various ligandscomprising the SHAL.

The SHAL and the effector can be attached by any of a number of meanswell known to those of skill in the art. Typically the effector isconjugated, either directly or through a linker (spacer), to the SHAL.

In one embodiment, the SHAL is chemically conjugated to the effectormolecule (e.g., a cytotoxin, a label, a ligand, or a drug or liposome,etc.). Means of chemically conjugating molecules are well known to thoseof skill.

The procedure for attaching an effector to a SHAL will vary according tothe chemical structure of the effector and/or the SHAL. The ligandscomprising the SHAL and/or the linker joining the ligands can contain avariety of functional groups; e.g., carboxylic acid (COOH), free amine(—NH₂), hydroxyl (—OH), thiol (—SH), and other groups, that areavailable for reaction with a suitable functional group on an effectormolecule or on a linker attached to an effector molecule to effectivelybind the effector to the SHAL.

Alternatively, the ligand(s) comprising the SHAL and/or the linkerjoining the ligands can be derivatized to expose or attach additionalreactive functional groups. The derivatization can involve attachment ofany of a number of linker molecules such as those described above forcoupling the ligands to each other.

In some circumstances, it is desirable to free the effector from theSHAL when the chimeric molecule has reached its target site. Therefore,chimeric conjugates comprising linkages that are cleavable, e.g., in thevicinity of the target site can be used when the effector is to bereleased from the SHAL. Cleaving of the linkage to release the agentfrom the antibody may be prompted by enzymatic activity or conditions towhich the conjugate is subjected, e.g., either inside the target cell orin the vicinity of the target site. When the target site is a tumor, alinker which is cleavable under conditions present at the tumor site(e.g., when exposed to tumor-associated enzymes or acidic pH) may beused.

In certain instances, the cleavable linker can be a peptide that can besubject to proteolysis. In certain embodiments, the cleavable linkercomprises a peptide having a recognition site for a protease.

A number of different cleavable linkers are known to those of skill inthe art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014. Themechanisms for release of an agent from these linker groups include, forexample, irradiation of a photolabile bond and acid-catalyzedhydrolysis. U.S. Pat. No. 4,671,958, for example, includes a descriptionof immunoconjugates comprising linkers which are cleaved at the targetsite in vivo by the proteolytic enzymes of the patient=s complementsystem. In view of the large number of methods that have been reportedfor attaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, drugs, toxins, and other agents to antibodies one skilled inthe art will be able to determine a suitable method for attaching agiven agent to an antibody or other polypeptide.

1) Conjugation of Chelates.

In certain preferred embodiments, the effector comprises a chelate thatis attached to an antibody or to an epitope tag. The cancer specificSHAL bears a corresponding epitope tag or antibody so that simplecontacting of the SHAL to the chelate results in attachment of the SHALto the effector. The combining step can be performed before the moietyis used (targeting strategy) or the target tissue can be bound to theSHAL before the chelate is delivered. Methods of producing chelatessuitable for coupling to various targeting moieties are well known tothose of skill in the art (see, e.g., U.S. Pat. Nos. 6,190,923,6,187,285, 6,183,721, 6,177,562, 6,159,445, 6,153,775, 6,149,890,6,143,276, 6,143,274, 6,139,819, 6,132,764, 6,123,923, 6,123,921,6,120,768, 6,120,751, 6,117,412, 6,106,866, 6,096,290, 6,093,382,6,090,800, 6,090,408, 6,088,613, 6,077,499, 6,075,010, 6,071,494,6,071,490, 6,060,040, 6,056,939, 6,051,207, 6,048,979, 6,045,821,6,045,775, 6,030,840, 6,028,066, 6,022,966, 6,022,523, 6,022,522,6,017,522, 6,015,897, 6,010,682, 6,010,681, 6,004,533, 6,001,329, andthe like).

III. SHALs that Inhibit Receptors, Enzymes and Other Biomolecules.

In certain embodiments, this invention provides SHALs that inhibit theactivity of enzymes, receptors, or the activity of other biomolecules.Typically such SHALS comprise ligands that bind to different sitesaround or near the active site or binding site of the enzyme orreceptor. In certain embodiments, the ligands are selected to bind to afirst pocket and a second pocket in the enzyme, receptor, or otherbinding protein where the first and second pocket flank opposite sidesof the active site or biding site of said enzyme, receptor, or otherbinding protein or the first pocket comprises or is located in activesite and second pocket is located nearby/adjacent to the first pocket oreither or both pockets are located in or sufficiently near the site usedby the protein, enzyme, or receptor for binding to another molecule suchthat the binding of a ligand in either or both pockets disrupts or blockthe binding of the enzyme, receptor, or other biomolecule to is cognateligand. When the SHAL is contacted with its target, it binds to thetarget effectively blocking the active site and/or binding site therebyin habiting the activity of the enzyme, receptor or other biomolecule.

IV) Pharmaceutical Compositions.

The SHALs, and/or chelates, and/or chimeric molecules of this invention(particularly those specific for cancer or other pathologic cells) areuseful for parenteral, topical, oral, or local administration (e.g.,injected into a tumor site), aerosol administration, or transdermaladministration, for prophylactic, but principally for therapeutictreatment. The pharmaceutical compositions can be administered in avariety of unit dosage forms depending upon the method ofadministration. For example, unit dosage forms suitable for oraladministration include powder, tablets, pills, capsules and lozenges. Itis recognized pharmaceutical compositions of this invention, whenadministered orally, can be protected from digestion. This is typicallyaccomplished either by complexing the active component (e.g., the SHAL,the chimeric molecule, etc.) with a composition to render it resistantto acidic and enzymatic hydrolysis or by packaging the activeingredient(s) in an appropriately resistant carrier such as a liposome.Means of protecting components from digestion are well known in the art.

The pharmaceutical compositions of this invention are particularlyuseful for parenteral administration, such as intravenous administrationor administration into a body cavity or lumen of an organ. Thecompositions for administration will commonly comprise a solution of theSHAL and/or chimeric molecule dissolved in a pharmaceutically acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carrierscan be used, e.g., buffered saline and the like. These solutions aresterile and generally free of undesirable matter. These compositions maybe sterilized by conventional, well known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example, sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate and the like. The concentration ofchimeric molecule in these formulations can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight andthe like in accordance with the particular mode of administrationselected and the patient's needs.

Thus, a typical pharmaceutical composition for intravenousadministration would be about 0.1 to 10 mg per patient per day. Dosagesfrom 0.1 up to about 100 mg per patient per day may be used,particularly when the drug is administered to a secluded site and notinto the blood stream, such as into a body cavity or into a lumen of anorgan. Actual methods for preparing parenterally administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington'sPharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa.(1980).

The compositions containing the present SHALs and/or chimeric moleculesor a cocktail thereof (i.e., with other therapeutics) can beadministered for therapeutic treatments. In therapeutic applications,compositions are administered to a patient suffering from a disease,e.g., a cancer, in an amount sufficient to cure or at least partiallyarrest the disease and its complications. An amount adequate toaccomplish this is defined as a “therapeutically effective dose.”Amounts effective for this use will depend upon the severity of thedisease and the general state of the patient's health.

Single or multiple administrations of the compositions may beadministered depending on the dosage and frequency as required andtolerated by the patient. In any event, the composition should provide asufficient quantity of the SHALs to effectively treat the patient.

It will be appreciated by one of skill in the art that there are someregions that are not heavily vascularized or that are protected by cellsjoined by tight junctions and/or active transport mechanisms whichreduce or prevent the entry of macromolecules present in the bloodstream

One of skill in the art will appreciate that in these instances, thetherapeutic compositions of this invention can be administered directlyto the tumor site. Thus, for example, brain tumors can be treated byadministering the therapeutic composition directly to the tumor site(e.g., through a surgically implanted catheter).

Alternatively, the therapeutic composition can be placed at the targetsite in a slow release formulation. Such formulations can include, forexample, a biocompatible sponge or other inert or resorbable matrixmaterial impregnated with the therapeutic composition, slow dissolvingtime release capsules or microcapsules, and the like.

Typically the catheter or time release formulation will be placed at thetumor site as part of a surgical procedure. Thus, for example, wheremajor tumor mass is surgically removed, the perfusing catheter or timerelease formulation can be emplaced at the tumor site as an adjuncttherapy. Of course, surgical removal of the tumor mass may be undesired,not required, or impossible, in which case, the delivery of thetherapeutic compositions of this invention may comprise the primarytherapeutic modality.

V. Kits.

Where a radioactive, or other, effector is used as a diagnostic and/ortherapeutic agent, it is frequently impossible to put the ready-for-usecomposition at the disposal of the user, because of the often poor shelflife of the radiolabeled compound and/or the short half-life of theradionuclide used. In such cases the user can carry out the labelingreaction with the radionuclide in the clinical hospital, physician'soffice, or laboratory. For this purpose, or other purposes, the variousreaction ingredients can then be offered to the user in the form of aso-called “kit”. The kit is preferably designed so that themanipulations necessary to perform the desired reaction should be assimple as possible to enable the user to prepare from the kit thedesired composition by using the facilities that are at his disposal.Therefore the invention also relates to a kit for preparing acomposition according to this invention.

Such a kit according to the present invention preferably comprises aSHAL as described herein. The SHAL can be provided, if desired, withinert pharmaceutically acceptable carrier and/or formulating agentsand/or adjuvants is/are added. In addition, the kit optionally includesa solution of a salt or chelate of a suitable radionuclide (or otheractive agent), and (iii) instructions for use with a prescription foradministering and/or reacting the ingredients present in the kit.

The kit to be supplied to the user may also comprise the ingredient(s)defined above, together with instructions for use, whereas the solutionof a salt or chelate of the radionuclide which can have a limited shelflife, can be put to the disposal of the user separately.

The kit can optionally, additionally comprise a reducing agent and/or,if desired, a chelator, and/or instructions for use of the compositionand/or a prescription for reacting the ingredients of the kit to formthe desired product(s). If desired, the ingredients of the kit may becombined, provided they are compatible.

In certain embodiments, the complex-forming reaction with the SHAL cansimply be produced by combining the components in a neutral medium andcausing them to react. For that purpose the effector may be presented tothe SHAL in the form of a chelate.

When kit constituent(s) are used as component(s) for pharmaceuticaladministration (e.g., as an injection liquid) they are preferablysterile. When the constituent(s) are provided in a dry state, the usershould preferably use a sterile physiological saline solution as asolvent. If desired, the constituent(s) can be stabilized in theconventional manner with suitable stabilizers, for example, ascorbicacid, gentisic acid or salts of these acids, or they may comprise otherauxiliary agents, for example, fillers, such as glucose, lactose,mannitol, and the like.

While the instructional materials, when present, typically comprisewritten or printed materials they are not limited to such. Any mediumcapable of storing such instructions and communicating them to an enduser is contemplated by this invention. Such media include, but are notlimited to electronic storage media (e.g., magnetic discs, tapes,cartridges, chips), optical media (e.g., CD ROM), and the like. Suchmedia may include addresses to internet sites that provide suchinstructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1. Creation of HLA-DR10 Specific SHALs

Preclinical and clinical studies have revealed that the epitopic region(unique region recognized by antibodies) on the beta subunit of HLA-DR10, and related HLA-DR major histocompatibility cell surface proteins,are particularly attractive targets for systemic radioisotopic therapyfor B-cell lymphomas and leukemias and provide other opportunities forcancer treatment and prevention. Although HLA-DR 10 has characteristicsin common with other B-cell surface proteins, like CD20, that make it asuitable target, it has disparate characteristics that we believeprovide great attractiveness.

In common with CD20 antigen, the HLA-DR 10 protein is located on thesurface of B lymphocytes, persists through B-cell differentiation, butdisappears during transformation of the lymphocyte to the plasma cellstage (Epstein et al. (1987) Cancer Res., 47: 830-840). The discreteLym-1 antigen epitope appears on committed B-cell precursors, but is notexpressed earlier during B-cell development. In addition, it is notgenerally found on T cells or other normal cells. Expression of class 2MHC molecules on B-cells is developmentally controlled. Early andpre-B-cells are class 2 mRNA negative and cannot be induced to expressclass 2 antigens. HLA-DR antigen is acquired during the late pre-B-cellstage. Because the basal level of class 2 expression on B-cells is about100 times lower than that found on malignant B-cell lines (Rose et al.(1996) Cancer Immunol Immunother., 43: 26-30; Rose et al. (1999) MolImmunol., 36: 789-797), this provides an explanation for the observationthat only 5 mg of Lym-1 antibody is needed to target extravascularmalignant lymphoma (DeNardo et al. (1998) J Clin Oncol., 16: 3246-3256),whereas 50 mg of CD22 and hundreds of mg of CD20 antibodies are requireddue to the density of CD22 and CD20 antigens on normal lymphocytes(Press (1999) Semin Oncol., 26: 58-65; Knox et al. (1996) Clin CancerRes., 2: 457-470). Treatment doses of iodine-131, copper-67, oryttrium-90 attached to small amounts of Lym-1 cures most mice with Rajixenografts (DeNardo et al. (1991) Antibody Immunoconj Radiophar., 4:777-785; DeNardo et al. (1997) Clin Cancer Res., 3:71-79), the humanBurkitt's malignant lymphoma cell line used as the immunogen to generateLym-1 (Epstein et al. (1987) Cancer Res., 47: 830-840). Similarly, theseradiopharmaceuticals, in Phase I/II trials in patients with B-cellnon-Hodgkin's lymphoma and a subset of patients with chronic lymphocyticleukemia, have induced a high and durable response rate, with frequentcomplete remissions and some long-term survivals when used as singleagent therapy. HLA-DR provides cell identification, and antigenicpeptides are displayed on HLA-DR. This may explain unusually longsurvivals in a subset of our patients with aggressive lymphoma in whoman idiotypic antibody cascade, including human polyclonal antibodiescytotoxic for Raji cells and Raji tumors, has been documented (DeNardoet al. (1998) Cancer Biother Radiopharm., 13: 1-12; Lamborn et al.(1997) Clin Cancer Res., 3: 1253-1260).

However, as well as these antibodies work, there is still a need toimprove upon them. The antibody is a macromolecule that penetratesvascular barriers and the tumor poorly and interacts with a variety orreceptors, which limits their selectivity as radioisotope carriers andadds to the adverse event profile. The immunogenicity of antibody-basedreagents can be minimized, but not eliminated, using “humanized”antibodies (Brown et al. (2001) Clin Lymphoma., 2: 188-90; Kostelny etal. (2001) Int J Cancer, 93: 556-65; Leonard et al. (2002) Semin Oncol.,29: 81-6; Lundin et al. (2002) Blood, 100: 768-73; Ligibel and Winer(2002) Semin Oncol., 29: 38-43). Immunogenicity may be avoided bycreating non-protein based reagents. Whole antibodies also exhibitappreciable reactivity (e.g., Fc interactions) with non-target cellsthat reduces selectivity and increases adverse events. Even smallimprovements in the targeting agent's selectivity can be used tominimize collateral damage and enhance the drug's therapeutic index.

Lym-1 is a murine IgG-2a monoclonal antibody (MAb) that selectivelybinds a protein highly expressed on the surface of malignant humanB-cells (Epstein et al. (1987) Cancer Res., 47: 830-840). We have shownthat a discrete epitope on HLA-DR 10 was the original antigen in Rajicells that generated the Lym-1 MAb, and this epitope is not shared byall HLA-DR subtypes (Rose et al. (1996) Cancer Immunol Immunother., 43:26-30; Rose et al. (1999) Mol Immunol., 36: 789-797). Our data suggestthat the critical Lym-1 binding residues are contained in the 19differences in amino acid sequence between the reactive HLA-DR 10 betasubunit and the unreactive, largely identical HLA-DR 3 and HLA-DR 52beta subunits. This serves as the basis for the selectivity of the Lym-1epitope or binding site among HLA-DR containing white blood cells, yetprovides the basis for the existence of this protein in virtually allpatients with malignant B-cells. Of the 19 residues comprising thecritical Lym-1 binding region, only the amino acids Q70 or R70, followedby R71 were found in all Lym-1 reactive specimens and were absent inLym-1 unreactive specimens. In many of the unreactive HLA-DR molecules,these two residues were often replaced by D70 and/or E71. The hypothesisthat the subtypes containing the putative critical Lym-1 bindingresidues (Q/R70-R71) would be most reactive has been confirmed in aseries of studies including extensive cytotoxicity assays conducted inlymphoblastoid cell lines of B and T cell type, incorporating 31 HLA-DRgenotypes (Rose et al. (1999) Mol Immunol., 36: 789-797). All thestrongly reactive cells expressed at least one Q/R70-R71-containingHLA-DR allele while none of the least reactive cell lines expressed thatsequence at position 70-71 of the beta chain. Cytotoxicity assays alsoshowed that the former were dramatically more affected than the latter(Id.). Although Lym-1 reacted with peripheral blood lymphocytes fromhealthy donors, the avidities were much lower, consistent with a lowerHLA-DR protein density on normal lymphocytes and the hypothesis thatunivalent rather than bivalent binding may occur, further explaining theselectivity of Lym-1 for malignant cells in patients with lymphoma(Id.). Thus, it seems that both the critical Lym-1 glutamine/arginineresidues and a threshold antigen density contribute to the selectivityof Lym-1 binding to malignant B-cells over normal lymphocytes. In anyevent, the data confirm that Lym-1 binds preferentially tolymphoblastoid cells over normal PBLs, thereby providing an attractivedifference from other malignant B-cell targeting proteins (Id.).

Applicants' experience with CMRIT has led us to appreciate thecomplexities of implementation and to realize that many patients withadvanced NHL are ineligible for BMT because of their disease andinsufficient marrow harvest. For these reasons and because of uniqueopportunities to dose intensify using novel approaches to developtargeting molecules that can dramatically improve the therapeutic index,we have developed high affinity ligands (SHALs) that mimic ¹³¹I-iodidein thyroid cancer. ¹³¹I-iodide in thyroid cancer, the prototype forradioisotopic molecular targeted systemic radiotherapy, has led to cureof otherwise incurable thyroid cancer because the ¹³¹I is rapidlytrapped and retained by the cancer or excreted in the urine, providing atherapeutic index approaching infinity and the opportunity to administeralmost unlimited radioisotope without significant toxicity.

We believe that small molecule SHALs can better fulfill the potential of“RIT”, and represent a natural extension of our ongoing translationalactivities involving HLA-DR as a target for radioisotopic carriermolecules to deliver systemic radiotherapy. As described below, we havesynthesized a number of bidentate SHALs and determined that at least oneof these SHALs binds to isolated HLA-DR10.

A) Development of a Computer Model of the Molecular Structure of theHLA-DR 10 Beta Subunit Containing the Region Shown to be Critical forLym-1 Antibody Binding to Malignant B Cells and Compare the Structurewith Other HLA-DR Molecules.

Crystal structures for four different closely related HLA-DR molecules(HLA-DR 1-4) have been determined previously and deposited in the PDBstructure database by others (Jardetzky et al. (1994) Nature,368:711-718; Bolin et al. (2000) J. Med. Chem., 43:2135-2148; Smith etal. (1998) J. Exp. Med., 188:1511-1520; Ghosh et al. (1995) Nature,378:457-462). Protein sequences for these four proteins, HLA-DR1,HLA-DR2, HLA-DR3 and HLA-DR4, were aligned with the HLA-DR10 sequenceand compared to identify both the locations of the variable amino acidsand those regions of the HLA-DR10 molecule containing the amino acidresidues that had been identified as the critical epitope of the Lym-1antibody (Rose et al. (1996) Cancer Immunol Immunother., 43: 26-30; Roseet al. (1999) Mol Immunol., 36: 789-797). This alignment revealed thatall five proteins exhibit such a high degree of sequence similarity(FIG. 8) that we were able to create a sufficiently accurate 3-D modelof the HLA-DR10 beta subunit by homology modeling and use thecoordinates of the model to screen for ligand binding using the programDOCK.

Two different approaches were used to create models of the HLA-DR10 betasubunit for use in ligand docking. The first approach used thecoordinates of the entire structure of HLA-DR3 as the template forcreating the homology model, and the nineteen amino acids that differedbetween HLA-DR3 and HLA-DR10 were mutated (changed) in the HLA-DR3sequence. The coordinates of the amino-terminal four amino acids, whichare present in HLA-DR10, HLA-DR1 and HLA-DR2 but absent in HLA-DR3, wereobtained from the HLA-DR1 structure and used to complete the model. Inthe second approach, a hybrid model was generated using the atomiccoordinates obtained from different segments of the HLA-DR 1, HLA-DR2and HLA-DR4 crystal structures. The particular segments of the three HLAcrystal structures used in the model were selected based on similaritiesin their secondary structural elements. Sequence-structure alignmentswere generated using the Smith-Waterman (Smith and Waterman (1981) J.Mol. Biol., 147:195-197), FASTA (Pearson (1991) Genomics, 11: 635-650),BLAST and PSI-BLAST (Altschul et al. (1997) Nucleic Acids Res., 25:3389-3402) algorithms, and the backbone of the model was createdautomatically using the AS2TS system (seehttp://sb9.llnl.gov/adamz/LGA/AL2TS/as2ts.html website). The coordinatesfor the amino terminal four residues of the structure were taken fromthe 1 seB crystal structure of HLA-DR1, residues 5-122 were obtainedfrom the 1aqd structure of HLA-DR1, residues 123-170 were taken from the1d5m HLA DR4 structure, and the remaining residues (aa171-193) wereobtained from the 1bx2 structure of HLA-DR2. The construction of theterminal regions and loops, amino acid insertions and deletions, andtemplate-model structure comparisons were performed using the LGAprogram developed at LLNL (see websitehttp://predictioncenter.llnl.gov/local/lga/lga.html). The majority ofthe side chain atom's coordinates were incorporated from the fourstructural templates (listed above) due to their high level of homology.The side chains in selected regions of the protein model were builtusing the SCWRL program (Id.). Energy minimization was performed on bothstructures to eliminate inappropriate side chain contacts and theresulting structures were “optimized” using molecular dynamics.

Analyses of the resulting models revealed the two approaches yieldedstructures that were remarkably similar. Extended molecular dynamicsruns appeared to provide little additional improvements. The results ofthe modeling revealed that the structure of the HLA-DR10 molecule iscomprised of two domains linked by a hinge with one of the Lym-1reactive residues, V85, positioned directly adjacent to the hinge (FIG.9).

The majority of the core of the relaxed structure of HLA-DR10, whencompared with the HLA-DR3 crystal structure, was found to be essentiallyidentical (FIG. 10). The other three amino acids that were observed toplay a role in Lym-1 binding, R70, R71 and A74 (A or E at this positionappears important for Lym-1 binding), are all located on the exposedsurface of a long alpha helix (FIGS. 9 and 10) located immediatelyadjacent to the hinge

B) Identification of Unique Sites on the Surface of HLA-DR10 within theLym-1 Epitope that can be Targeted for Ligand Binding.

Solvent accessible surfaces of the HLA-DR10 protein and the crystalstructure of HLA-DR3 were calculated using the atomic coordinatesobtained for HLA-DR3 from the Protein Data Bank and our HLA-DR10 model.The site surrounding the three key amino acids in the Lym-1 epitope(within 6 Å) were examined and compared. As shown in FIG. 11, thesethree amino acid changes in the HLA-DR10 sequence (Q70R, K71R, and R74A)change both the charge distribution and topography of the protein'sexposed surface in this region.

A program developed to identify “pockets” on the surface of the protein(SPHGEN) was used to identify potential cavities that could be targetedfor ligand binding. Details of the programs used are described inExample 2. Two adjacent pockets (cavities) in the modeled HLA-DR10surface were selected as appropriate sites for ligand binding (FIG. 12),based on their proximity to each other, the three Lym-1 reactiveresidues, and the uniqueness of the amino acids lining the pocket. Anexamination of the crystal structures of the other four HLA-DR moleculesalso showed that pockets exist at or in the near vicinity of these sitesin each HLA-DR molecule, but as expected (based on the amino acidsequence differences in the peptide chains in this region of the HLA-DRstructure) the pockets present on each HLA-DR differ in size, shape, anddistance separating them. Since the amino acid “landscape” surroundingthese two sites differs significantly in HLA-DR3 and HLA-DR10, dockingruns performed at both sites would be expected to identify ligands thatbind selectively to the pockets on HLA-DR10 but not to HLA-DR3. Thesesites, identified as Site 1 and Site 2 and shown filled with red andblue spheres in FIG. 12, flank both sides of the most important aminoacid in the Lym-1 epitope, R70. A third unique site, which can betargeted as a backup if suitable ligands cannot be identified that bindto Site 1 or Site 2, has also been located and characterized.

C) Computationally Screening a “Virtual” Ligand Library to IdentifySmall Molecules that May Bind to Specific Pockets on HLA-DR10Encompassing the Lym-1 Epitope.

The program DOCK (UCSF) was used to perform a “virtual” screen of theAvailable Chemical Directory database of small molecules to identify thetop ranked 1,000 molecules predicted to bind in the two unique pocketsidentified as Site 1 and Site 2 (FIG. 12). Details of the computationaldocking procedures are described in Example 2.

The top ranked 2,500 molecules were then visually inspected to selectdown to thirty-five molecules for experiment binding assays (Table 5).This final selection process was based on chemical properties includinghydrophobic interactions, hydrogen bonding, and molecular size, and wellas practical criteria including commercial availability and cost, easeof synthetic linkage, the overall structural diversity of the set ofmolecules.

TABLE 5 Ligands predicted to bind to Site 1 on the beta subunit ofHLA-DR10 by computational docking. No. Compound 1.7-Amino-4-chloromethylcoumarin, glycyl-l-proline amide, hydrochloride 2.5-([4,6-Dichlorotriazin-2-yl]amino)-fluorescein hydrochloride 3.2-[2-[3-Chloro-5-(trifluoromethyl)-2-pyridinyl]carbohydrazonoyl]benzenecarboxylic acid 4.4-[[2-(4-Cyano-3-phenyl-5-isoxazolyl)vinyl]amino]benzenecarboxylic acid5. 4-[2-(2,4-Dichlorophenyl)hydrazino]-4-oxo-2-phenyl-2-butenoic acid 6.3-(2-[[3-Chloro-5-(trifluoromethyl)-2-pyridinyl]oxy]anilino)-3-oxopropanoicacid 7. 3-[(4-Chlorobenzyl)thio]imidazo[1,5-a]pyridine-1-carboxylic acid8. 2-[([1,1′-Biphenyl]-4-ylamino)carbonyl]benzoic acid 9.Bis[4-(3-aminophenoxy)phenyl]sulfone 10.4,4′-Bis(4-aminophenoxy)biphenyl 11. 5(6)-Carboxytetramethylrhodaminen-succinimidyl ester 12.1,4-Phenylenebis[[4-(4-aminophenoxy)phenyl]methanone] 13.H-Tyr(Br-Z)-OEt 14. ′7-Amino-4-chloromethylcoumarin, 1-alanyl-l-prolineamide, hydrochloride 15. ′5-(N′-[2-aminoethyl]thioureidofluorescein) 16.Achatin I, Ammonium salt 17. Fmoc-Asp(OBzl)-OH 18. Fmoc-Bip-OH 19. MenaiH535 20. 2′-Methoxy-5′-methyl-3,4,5,6-tetrachlorophthalanilic acid 21.4-Dimethylaminoazobenzene-4′-sulfonyl-l-valine 22. Bigchap 23.Arg-gly-asp-thr (SEQ ID NO: 25) 24.n-Allyl-2-[(1-benzyl-2-oxo-1,2-dihydro-3-pyridinyl)carbonyl]-1-hydrazinecarbothioamide 25.n′-Methoxy-n-[7-(4-phenoxyphenyl)[1,2,4]triazolo[1,5-a]pyrimidin-2-yl]iminoformamide 26.n-[[6-(4-Chlorophenoxy)-3-pyridyl]carbonyl]-n′-[3-(trifluoromethyl)phenyl]urea27. n-[[6-(4-Chlorophenoxy)-3-pyridyl]carbonyl]n′-(4-chlorophenyl)urea28. n,n′-Diphenylbenzidine 29. Rcl s16,963-3 30.N,N′-bis-(4-amino-2-chloro-phenyl)-terephthalamide4-[[5-(trifluoromethyl)pyridin-2-yl]oxy]phenyl N-phenylcarbamate 31. Methidiumpropyl ethylenediametetraacetic acid 32.N-(4-[[3-Chloro-5-(trifluoromethyl)-2-pyridinyl]methyl]phenyl)-4-iodobenzenecarboxamide 33.2-(4-Chlorophenyl)-2-[6-[(4-chlorophenyl)sulfanyl]-3-pyridazinyl]acetamide34. 6-Chloro-n4-(4-phenoxyphenyl)-2,4-pyrimidinediamine 35.4-Amino-2-anilino-5-benzoyl-3-thiophenecarbonitrile

This procedure was repeated for identification of thirty-five potentialbinders for Site 2. Table 6 lists those ligands predicted by moleculardocking to bind to Site. 2.

TABLE 6 Ligands predicted to bind to Site 2 on the beta subunit ofHLA-DR10 by computational docking. 1. Leu-enkephalin 2.4-[4-(4-Chlorobenzyl)piperazino]-3-nitrobenzenecarboxylic acid 3.Beta-casomorphin (1-2) 4. L-Aspartic acid,alpha-(4,5-dimethoxy-2-nitrobenzyl) ester, hydrochloride 5. Cefadroxil6. 3,3′,5-Triiodo-dl-thyronine 7. H-Glu(anilide)-OH 8. H-Trp-phe-OH 9.Glycylglycyl-D,L-phenylalanine 10. Thymopoietin II (33-36) 11.Thiophosphoric acid S-(3-(3-amino-propylamino)-propyl) ester, di-hydrate12. Dynorphin A (13-17), Porcine 13. 1-Alanyl-l-alanyl-l-tryptophan 14.Asp-arg-val-tyr (SEQ ID NO: 26) 15. A-VI-5 16. Glu-thr-pro NH₂ 17.(D-ala2)-Beta-casomorphin (1-5) (bovine) 18. Tyr-D-ala-gly-phe-D-metacetate salt (SEQ ID NO: 27) 19. Arg-gly-asp-thr (SEQ ID NO: 28) 20.N-Alpha,n-omega-di-cbz-l-arginine 21. Asp-lys acetate salt 22.(+)-Allo-octopine 23. Sodium7-[(2-amino-2-phenylacetyl)amino]-3-methyl-8-oxo-5- thia-l-azabiccyclo[4.2.0]oct-2-ene-2-carboxylate 24. Val-ile-his-asn (SEQ ID NO: 29)25. 2-Amino-8-(diphenylphosphinyl)-octanoic acid 26. Glu-His-Pro NH₂ 27.Bapaba 28. H-glu(lys)-OH 29. Bis-boc-l-arg 30. H-arg(mtr)-OH 31.4-Aminomethyl-L-phenylalanine boc 32. H-met-met-OH 33. H-trp-ile-OH 34.N-alpha benzoyl-arginine-4-amino benzoic acid 35. (Thr46)-osteocalcin(45-49) (human)

As a result of the computational docking, 30 compounds from both theSite 1 and Site 2 lists were experimentally screened by NMR. Elevencompounds were found to bind to HLA-DR10, giving a successful hit rateof 37%. These ligands are listed in Table 7.

TABLE 7 NMR screened ligands that were found to bind to HLA-DR10.Ligands are separated by computationally predicted docking sites Site 1:Site 2: 5(6) carboxytetramethylrhodamine-n- N-alpha benzoyl-arginine-succinimidyl ester 4-amino benzoic acid Methidiumpropyl EDTA5-leu-enkephalin (YAGFM) Deoxycholic acid N alpha N omegadicarbobenzoxyarginine FMOC-aspartic acid(O-benzyl)-OH Angiotensin II(DRVY) 4-dimethylaminoazobenzene-4′-sulfonyl-L- Bis-BOC-L-argininevaline 4-[[5-(trifluoromethyl)pyridin-2- yl]oxy]phenyl N-phenylcarbamate

From Table 7, 5 synthetic high affinity ligands (SHALs) have beensynthesized, containing different sets of the Site 1 and Site 2 ligands.Three of these molecules (FIG. 13A), all containing the ligand pairsdeoxycholate and 5-leu-enkephalin, have been shown to bind to isolatedHLA-DR10. None of these three SHALs bind to albumin or streptavidin. Thefirst of the three to be tested more extensively, JP459B (FIG. 13B), hasbeen determined to bind to HLA-DR10 with a Kd=23 nM using SurfacePlasmon Resonance. Using Raji membrane extracts, this SHAL competed withLym-1 for binding to HLA-DR10. Binding studies performed with live cellsshowed JP459B bound to Raji human lymphoma cells, but not normal DU145,LnCAP or 22RV cells (FIGS. 17E-H). Experiments using frozen humanlymphoma tissue sections also demonstrated JP459B binding to large celllymphoma, exuberantly, but less to small cell lymphoma cells (FIGS.17A-D). Similar results were obtained with Lym-1 antibody. Moreextensive testing with normal and tumor tissue arrays is in progress.

In subsequent screenings additional ligands have been shown to bind toHLA-DR10. These are shown in Table 8.

TABLE 8 Additional ligands that bind to HLA-DR10. ligand ID species 113,3′,5-Triiodo-dl-thyronine (Predicted Site 2) (TI) 72-(4-Chlorophenyl)-2-[6-[(4-chlorophenyl)suflfanyl]-3- pyridazinyl]acetamide (12F) 9 4-Amino-2-anilino-5-benzoyl-3-thiophenecarbonitrile(5K) 8 6-Chloro-n4-(4-phenoxyphenyl)-2,4-pyrimidinediamine (7L) 6N-(4-[[3-Chloro-5(trifluoromethyl)-2-pyridinyl]methyl]phenyl)-4-iodobenzenecarboxamide (6J)

In addition 1,4-phenylenebis[[4-(4-aminophenoxy)phenyl]methanone]precipitated onto the target protein.

Example 2. Computational Methods for Use in the Creation of SHALS A)Overview of the Roles and Methods of Molecular Simulations

As described herein, molecular modeling can be used to initiallyidentify ligands for use in the construction of SHALs and/or for theoptimization of SHALs. At present there is no single molecular modelingmethodology that can be used to model target molecules, screen forbinding ligands, simulate binding of a polydentate SHAL and predictoptimal SHAL structure. A number of well established modeling methods,however can be used to facilitate these tasks as described herein.

Starting at the highest level, the prediction of the tertiary structurestarget molecules (e.g., protein cancer markers) are typically predictedusing highly empirical methods based on primary sequence homology toproteins with experimentally known structure. The accuracy of theseso-called homology-based protein structure prediction methods depend onthe availability of homologous protein structures and the expertise ofthe individual modeler. To identify small molecules (ligands) thatspecifically bind into protein pockets, computational “docking” can beemployed as described herein. Docking uses a relatively simple empiricalforce field to describe the ligand-protein interaction and can thereforebe used to rapidly screen 100,000's of possible ligands. To determinepreferred macromolecular conformations and interactions, classicalmolecular dynamics can used, which models the molecules using empiricalball-and-spring force fields. Finally, for the precise prediction ofsmall molecule structures and interactions, one can computationallysolve the quantum mechanical equations describing the electrons andnuclei within the molecules. This so-called first principles approachcan either be used to determine the structures and energies of static“snapshots” of the molecules or to simulate the atomic motions of themolecular systems. The former approach, is referred to as ab initioquantum chemistry while the latter approach is called first principlesmolecular dynamics (in contrast to classical molecular dynamics) andconstitutes a nearly exact simulation of nature.

B) Homology-Based Protein Structure Predictions.

The basic concept of homology-based protein structure prediction relieson the observation that structural features of proteins are conservedduring evolution to a much higher degree than their sequences, andtherefore proteins related even by distant sequence similarity can beexpected to have similar 3D structures (Chothia and Lesk (1986) EMBO J.,5: 823-826). Thus, once a three-dimensional structure is determined forat least one representative of a protein family, models for other familymembers can be derived using the known structure as a template.Homology-based protein modeling consists of four major steps: findingknown structures related to the protein sequence to be modeled, aligningthe sequence with these structures, building a three-dimensional model,and assessing the model (Marti-Renom et al. (2000) Annu Rev BiophysBiomol Struct., 29: 291-325).

Homology-based protein structure prediction (also referred to ascomparative modeling) produces an all-atom model of a sequence based onits alignment with one or more related protein structures. Building ofthe three-dimensional model itself includes either sequential orsimultaneous modeling of the core of the protein, loops and side chains.FIG. 15 illustrates a flowchart describing major steps in homology-basedprotein modeling.

The accuracy of a model, built using comparative modeling technique,usually is related to the percentage of sequence identity with thestructure on which it is based. High-accuracy comparative models arebased on more than 50% sequence identity to their templates. They tendto have about 1 Å root-mean-square (RMS) error for the main chain atoms.Such accuracy is comparable with medium resolution nuclear magneticresonance (NMR) structure or low resolution X-ray structure. The errorsin such cases are usually limited to mistakes in side chain rotamerassignment, small shifts or distortions in the core main chain regions,and occasionally larger errors in loops.

One general modeling approach will be similar to that successfully usedearlier to model both high and low homology target proteins (Venclovaset al. (1999) Proteins-Structure Function and Genetics, 73-80). Sinceour modeling objects (e.g., cancer markers such as HLA-DR10) can havehigh sequence homology (>50% sequence identity) we can rely on pairwisesequence comparison of modeling target (query) with the proteins ofknown structures (from the Protein Data Bank (PDB)) to identify theclosest structural templates. To do this, a sensitive Smith-Watermanpairwise sequence comparison algorithm (Smith and Waterman (1981) J.Mol. Biol, 147: 195-197) implemented in the SSEARCH program (Pearson(1991) Genomics 11: 635-650) can be used. At the high level of sequencehomology structure alignment for the conserved structural regions can beused directly in model-building.

When a number of structural templates of comparable similarity areavailable one can use MODELLER, a comparative modeling program capableof automatically combining a number of template structures to betterrepresent the structure of the query. Where critical regions are presentin the target molecule, special care can be taken in assigningconformations to these regions. The candidate conformations for theseregions can be produced by searching a database of homologous structuresfor the fragments of identical length that also satisfy the stericconstraints for these regions. Both sequence similarity and structuralcontext near the region can be taken into account in selecting theactual conformation. Side chains within the model can be positionedusing a backbone-dependent rotamer library (Bower et al. (1997) J. Mol.Biol. 267: 1268-1282).

Assessment of the obtained models can be done using several techniques.One of these, ProsaII (Sippl (1993) Proteins, 17: 355-362; Aloy et al.(2000) J Comput Aided Mol Des. 14: 83-92), which is used to detecterrors in protein structures, creates an energy profile along thesequence of the protein. The regions that are assigned high energyvalues by ProsaII often serve as good indicators of errors inrepresenting the structure of these particular regions. For the detailedchecks of modeled structures, the structure verification module of theWHATIF program (Vriend (1990) J. Mol. Graph 8: 52-56) can be used alongwith visual inspection. If these assessments of model quality identifyany problems in the modeled structure, appropriate steps (such as loopassignment or side chain positioning) will be repeated in an iterativemanner until an acceptable quality three-dimensional model is obtained.

Using these methods a computer model of the molecular structure of theHLA-DR 10 beta subunit containing the region shown to be critical forLym-1 antibody binding to malignant B cells was developed and thestructure was compared with the structure with other HLA-DR molecules(see Example 1, supra.). This model was used to develop HLA-DR10specific SHALs as described herein.

C) Computational Docking

Computational methods such as docking have been used to speed up theprocess of drug discovery and inhibitor design by screening largenumbers of molecules and predicting whether or not they bind into theactive sites of target proteins (Desjarlais et al. (1990) Proc. Natl.Acad. Sci. U.S.A., 87: 6644-6648; Mao et al. (1998) Bioorganic andMedicinal Chem. Letts., 8: 2213-2218; Olson and Goodsell (1998)Environmental Res., 8: 273-285; Rutenber et al. (1993) J. Biol. Chem.,268: 15343-15346). These efforts have met with moderate success in thedesign of new drugs effective against HIV proteins critical forinfection and transmission of the disease

In certain embodiments, this approach is generally useful as a firststep in the identification of ligands (binding moieties) that usuallybind to the target molecule(s) in the micromolar range. Detailedprotocols for docking methods using SPHGEN and DOCK have been describedin the literature. For example, these methods have been used to identifyligands that bind to specific sites on the targeting domain of tetanusneurotoxin (Cosman et al. (2002) Chem Res Toxicol 15: 1218-1228;Lightstone et al. (2000) Chem. Res. Toxicol., 13: 356-362).

The program DOCK 4.0 was used to computationally screen the AvailableChemical Directory (˜300,000) of small molecules to identify the topranked 2,500 molecules predicted to bind to the identified Site 1 andSite 2 (FIG. 12).

Computational docking can be thought of as a three-step process: 1) siteidentification of the protein surface; 2) docking of ligands into theidentified binding site; and 3) scoring and the ranking of the ligands(Halperin et al. (2002) Proteins: Structure, Function, and Genetics, 47:409-443). For site identification, the solvent accessible surface of thetarget protein is generally calculated. Using the program SPHGEN, autility in DOCK (Moustakas and Kuntz (2002). DOCK5.0 (San Francisco,UCSF)), concave pockets on the protein surface were identified byfilling the pockets with different sized radii spheres. Essentially,this calculates the volume of the pocket. The surface of the protein mayhave anywhere from thirty to hundreds of pockets based on the size andshape of the protein. Once these pockets were identified, visualinspection of the pockets identified the binding site based on the sizeof the pocket and the available experimental evidence, such as knownamino acids involved in binding or catalysis. The chosen binding pocketwas then used in the subsequent docking procedure.

Docking studies identify small molecules that might bind specifically tothe chosen binding site on the protein. The DOCK 5.0 program screens adatabase of compounds on the computer and predicts which molecules willlikely bind tightly to the binding site. This procedure is illustratedin FIG. 16 We used the Available Chemicals Directory (ACD) from MDL asour database of compounds to screen.

The database was prepared by prefiltering to remove soaps and dyes.After the partial charges for the compounds were calculated by usingGasteiger-Marsili charges (Gasteiger and Marsili (1978) TetrahedronLetts., 34: 3181-3184; Gasteiger and Marsili (1980) Tetrahedron 36:3219-3288; Gasteiger and Marsili (1981) Organic Magnetic Resonance15:353-360) in Sybyl, the database was divided by total compound charge,and compounds with formal charge >±3 are filtered out. Also, compounds<10 and >80 heavy atoms (not hydrogens) were removed to focus oncompounds within the size range for lead (preliminary) drug compounds.This prefiltering made the database more efficient and eliminatedunnecessary calculations on compounds known to either never bind or bindindiscriminately. To simulate a flexible docking technique, 20 uniqueconformations were generated for each compound in the database. Each ofthese conformations was then rigidly docked into the binding site.Different orientations within the binding site were examined for each ofthe conformations of each of the ligands. All compounds were scored byenergy minimization where the intermolecular van der Waals andelectrostatic terms are derived from AMBER (Weiner (1984) J. Am. Chem.Soc., 106: 765-784). Though the molecules are ranked based on thescores, the scoring function does not predict the binding affinities.

The top ranked 2,500 molecules were then visually inspected to selectdown to thirty-five molecules for experiment binding assays as describedin Example 1. Ligands were selected and bidentate SHALs were constructedand tested as described in Example 1.

The binding of lead compounds to the target can be improved by severalorders of magnitude by using multiple (2-3) compounds linked together.For the inhibitor to be effective, it needs to recognize specificallythe target protein and bind with high affinity.

D) Quantum Chemical Calculations

A wide variety of chemical simulation methods have been developed,ranging from empirical ball-and-spring type molecular mechanics modelsto ab initio (first principles) quantum chemical methods that calculateapproximate solutions to the exact quantum mechanical equationsdescribing the electrons and nuclei. Typically, the choice of methodsinvolves trade-offs between accuracy, size of the chemical system, andcomputational cost. These modeling methods can be broadly divided intomolecular dynamics methods that simulate the time evolution of chemicalprocesses and static methods that predict time-independent molecularproperties such as the lowest energy configuration of a molecule or theenergy of a chemical reaction. One can use all three molecular modelingmethods described below: ab initio quantum chemistry, classicalmolecular dynamics and first principles molecular dynamics in the designand optimization of SHALs as described herein.

1. Ab Initio Quantum Chemistry (QM)

Ab initio quantum chemistry involves computing approximate solutions tothe exact non-relativistic Schroedinger equation describing a molecularsystem (Jensen (1999) Introduction to Computational Chemistry, New York,John Wiley and Sons). In principle these methods can predict theproperties of any chemical system to arbitrary accuracy, but in practicethe computational cost limits the accuracy of these methods and the sizeof the molecular systems to which they can be applied. Nevertheless, abinitio quantum chemical calculations are routinely applied to calculateaccurate structures and reaction energies for molecular systemsincluding up to hundreds of atoms.

There is a hierarchy of different ab initio quantum chemical methodsinvolving increasingly accurate mathematical descriptions of theelectronic wave function—the mathematical description of thedistribution of electrons around the nuclei of a molecule (Id.).Application of quantum chemistry typically requires the choice of boththe description of the electron-electron interactions (level of theory)and the spatial flexibility of the electrons (basis set). A fairly newclass of methods called Density Functional Theory (DFT) has beendeveloped that includes empirical parameterizations of theelectron-electron interactions, and often provides accuracy comparableto the earlier high-level quantum chemical methods (such as CoupledCluster methods), but with a much lower computational cost. The DFTmethods are usually denoted by the empirical electron-electron“functional” employed. Two widely used DFT functionals are the Becke3-parameter hybrid exchange functional (Becke (1993) J. Chem. Phys. 98:5648-5652) and the Lee-Yang-Parr gradient corrected electron correlationfunctional (Lee et al. (1988) Chemical Physics 123: 1-25). These havebeen widely demonstrated to yield accurate chemical structures andreaction energies for most molecules when used with sufficient basissets (Jensen (1999) Introduction to Computational Chemistry, New York,John Wiley and Sons).

The quantum chemical simulations described herein are used to studychemical processes that occur in the immediate extracellularenvironment. The quantum chemical methods described above typicallydescribe only an isolated (usually described as “gas-phase”) moleculeand therefore do not include the chemical environment, such as solventmolecules and counterions, which frequently is critical to the structureand energetics of biological molecules. Explicitly including thesurrounding water molecules and counter ions is usually notcomputationally practical; however, several methods have been developedwithin the quantum chemistry approach for effectively including theeffects of solvent interactions. Typically these methods model thesolvent as a continuous medium that polarizes in response to the quantumchemically derived charges. Although there are many situations whereexplicit inclusion of the solvent is necessary, these so-calledpolarizable continuum models have proven reasonably accurate inpredicting solvent-phase chemical properties including total solvationenergies and acid constants (Schüürmann et al. (1998) J. PhysicalChemistry A 102: 6706-6712; Tran and Colvin (2000) J. MolecularStructure, Theochem 532: 127-137).

The Langevin dipole method of Warshel is related to these polarizablecontinuum models, but includes a more realistic representation of thepolar solvent. The Langevin dipole method models the solvent as a largeset of polarizable dipoles on a fixed three-dimensional grid (Luzhkovand Warshel (1992) J. Computational Chemistry 13: 199-213). Thisapproach has recently been parameterized for use with ab initio derivedsolute charges and shown to yield solvation energies for neutral andionic molecules comparable or better than PCM methods described above(Florian and Warshel (1997a) J Am Chem Soc., 119: 5473-5474).

2. First Principles Molecular Dynamics (FPMD)

By combining the forces determined directly from a QM method to drivethe classical motion of all the atoms in a simulation, one can achievethe accuracy of quantum mechanics (QM) with the advantages of classicalmolecular dynamics. This approach became computationally feasible withthe development of a new technique based on density functional theory(DFT) (Kohn Sham (1965) Physical Review 140: A1133) that treatselectronic degrees of freedom at the same time as the nuclear equationsof motion (Car and Parrinello (1985) Physical Review Letters 55:2471-2474; Galli and Parrinello (1991) Pp. 283-304 In: ComputerSimulation in Materials Science, The Netherlands, Kluwer AcademicPublishers). Since the method employs QM theory to describe the entiresystem, it is often referred to as first principles molecular dynamics(FPMD). In the typical implementation of FPMD, only the chemicallyactive valence electrons are explicitly described with an expansion in aplane-wave basis, while the chemically inert core electrons arerepresented by pseudopotentials (Galli and Pasquarello (1993) Pp.261-313 In: Computer Simulation in Chemical Physics, D. J. Tildesley,ed. Dordrecht, Kluwer; Yin and Cohen (1982) Physical Review B (CondensedMatter) 25: 7403-7412). Because the pseudopotentials are transferable bydesign, this method does not require reparameterization when new systemsare studied. In addition, the use of a plane wave basis set naturallylends itself to the application of periodic boundary conditions, so themethod is well suited for modeling systems in the condensed phase. Thismethod, combined with several other computational improvements (Gygi(1993) Physical Review B 48: 11692-11700; Hutter et al. (1994)Computational Materials Science 2: 244-248; Payne et al. (1992) Rev.Modern Physics 64: 1045-1097), has been instrumental in solving theproblem of integrating QM and MD.

The first applications of FPMD simulations were limited to small systemssuch as silicon (Car and Parrinello (1985) Physical Review Letters 55:2471-2474; Stich et al. (1989) Physical Review Letters 63: 2240-2243).As these methods have been continuously improved upon, and advancedcomputational resources have become available (such as the DOE teraflopscale supercomputers) it is now possible to investigate smallbiochemical systems containing several hundreds of atoms for picosecondtimescales (Carloni and Alber (1998) Perspectives in Drug Discovery andDesign 9/11: 169-179; Pantano et al. (2000) J. Molecular Structure(Theochem) 530: 177-181; Rovia and Parrinello (2000) InternationalJournal of Quantum Chemistry 80: 1172-1180). For example, we haverecently simulated the conformational dynamics of a small chemical modelof the DNA backbone in solution. As the number of systems that have beeninvestigated with this new approach increases, it is becoming clear thatthe increased computational expense is repaid in the form of extremelyaccurate structural and dynamical properties. In particular, suchmethods potentially allow for very accurate dynamical simulations ofchemical phenomena including chelator-metal ion interactions andenzyme-catalyzed reactions.

E) Classical Molecular Dynamics (MD)

Classical molecular dynamics can be used to in identifying the exactorientation of the ligands in the in the binding sites of the targetmolecule(s) (e.g. HLA-DR 10 binding sites (Site 1 and Site 2)). Thisinformation can be used in designing the multivalent ligands to carryradioisotopes selectively to the target molecule and/or to cellsdisplaying the target molecule.

In particular, this structural data helps identify which functionalgroups on the ligand(s) can be used to synthetically attach the linker.For all ligands that are experimentally verified to bind to the target,classical molecular dynamics can be performed on the ligand in thetarget binding sites to determine conformation and orientation of theligand and the specific interactions mediating the binding specificity(e.g., hydrogen bonds, electrostatic and Van der Waals interactions). Incertain embodiments, the molecular dynamics simulations will include thetarget and the ligands solvated in a periodic water box. For eachligand, 500 ps to several nanosecond simulations can be performed usingmultiple starting orientations.

An important component of the polydentate SHALs of this invention is themolecular linker between the individual ligands (binding moieties)comprising the SHAL. Typically this linker adopts an aqueous-phaseconformation (or set of conformations) that holds the two (or more)ligands at appropriate distances to efficiently bind into their bindingsite on the target surface. To assist in designing this linker classicalmolecular dynamics can be performed on the linker molecular alone andthe linker bound to specific ligand compounds.

The program CHARMM can be used to perform classical molecular dynamicssimulations on various linkers. For example, simulations performed withPEG linkers can utilize a different number of PEG units (4, 6, and 8) inthe molecules. The starting structures for all four simulations can havethe molecules in a fully extended conformation. These extended moleculescan be solvated in water boxes and sodium ions were added to each waterbox to neutralize the systems. These solvated systems were heated toe.g., to 300 K and allowed to equilibrate for 200 picoseconds. Typicallysimulations can be run at constant temperature (NVT ensemble) andelectrostatic interactions treated by particle mesh Ewald (PME)summation.

Initially the two compounds to be linked can be visually examinedtogether bound to the protein as determined by docking and moleculardynamics using computer graphics. For example, a polyethylene glycol(PEG) linker will be built using molecular drawing software (AMPAC)between the two compounds. This structure can then be simulated usingclassical molecular dynamics in a periodic water box, e.g., as describedabove for several nanoseconds. One can analyze the resulting data on thedynamical motions to measure the average ligand-ligand distance andrelative ligand orientation. This data can be compared with the distancebetween the ligands and relative orientation on the target 10 surface todetermine the optimal length for the linker.

Molecular dynamics simulations can also be used to investigate a numberof other properties on the overall SHALs to optimize their therapeuticeffectiveness. In particular molecular dynamics simulations can be usedto investigate a number of modifications including the chemicalstructure of the linker itself, and the two ligands at each end. Thegoal of these simulations will be to identify likely effects of suchmodifications on target binding efficiency and specificity prior toexpensive synthetic modifications.

Molecular dynamics methods use an empirically derived classical forcefield to simulate the motion of each atom in a chemical system. Thismethodology is highly developed for the simulation of nucleic acids,(Beveridge and McConnell (2000) Curr. Opinion in Structural Biology 10:182-196) and proteins (Brooks et al. (1983) J. Computational Chemistry4: 187-217; Cheatham and Brooks (1998) Theoretical Chemistry Accounts99: 279-288; Doniach and Eastman (1999) Curr. Opinion in StructuralBiology 9: 157-163). Typical published molecular dynamics simulationsinvolve 10-100,000 atoms (including both the biomolecules beingsimulated and a surrounding shell of water and counter ions) which aresimulated for multi-nanoseconds of time, with the largest publishedsimulation being a 1 microsecond simulation of a small protein (Duan andKollman (1998) Science 282: 740-744). The multinanosecond time scale isthought sufficient to capture structural relaxation and solventreorganization, and is long enough in some cases to simulate transitionsbetween different macromolecular conformations (Cheatham and Kollman(1996) J. Mol. Biol., 259: 434-444; Yang and Pettitt (1996) J. PhysicalChemistry A 100: 2564-2566).

A molecular dynamics simulation of polyethylene glycol (PEG) hasrecently been published that is relevant to the design of PEG linkersfor SHALs. Heymann and Grubmuller used classical molecular dynamics todescribe the conformational and elastic properties of individual PEGchains (Heymann and Grubmuller (1999) Chemical Physics Letters 307:425-432; Young and Lovell (1992) Introduction to Polymeres, New York,Chapman and Hall). They simulated a PEG 18-mer (˜1 kDalton molecularweight) in the aqueous phase (solvated by 1539 water molecules) and inthe gas-phase (to approximate solvation in a non-polar solvent such ashexadecane). They found that in the gas-phase the PEG rapidly collapsedto a compact structure with no local structure, as measured by thedegree to which the PEG had a helical local structure. In water, the PEGbehaves very differently. It does show a reduction in the radius ofgyration compared to the fully extended structure, but retains a markeddegree of helicity and therefore some degree of stiffness. Thesesimulations indicate that the local stiffening of the PEG structure iscaused by water molecules that form hydrogen bond bridges betweensuccessive oxygens in the PEG chain. They further simulated thestretching of the PEG chain with a range of forces from 0 to 500picoNewtons (this mimics experimental studies with Atomic ForceMicroscopes). They find good agreement in their predicted force versusextension curves with values recently measured in a single PEG molecule(Oesterhelt et al. (1999) New Journal of Physics 1: 6.1-6.11).

These results demonstrate that classical molecular dynamics simulationsof PEG can accurately reproduce complex properties such as theforce/extension curves and strongly supports the accuracy of theproposed PEGylated scaffold simulations. Although the PEGylated scaffoldcurrently in use (13.6 kDalton molecular weight) is considerably largerthan the PEG 18-mer simulated by Heymann and Grubmiller, it is wellwithin reach of routine molecular dynamics simulations.

Molecular dynamics simulations can readily be performed with the CHARMMsoftware package (Brooks et al. (1983) J. Computational Chemistry 4:187-217) using the version 22 parameter set (MacKerell et al. (1998) J.Physical Chemistry B 102: 3586-3616). Analysis can be performed usingthe analysis tools distributed with CHARMM and VIVID, a graphicalmolecular dynamics analysis tool (Humphrey et al. (1996) J. MolecularGraphics 14: 33-38).

The steps in a typical setup and simulation runs are as follows:

A. Preliminary Setup.

1. Calculation of partial charges for atom types not included in CHARMMforce field. Model compounds containing the unparameterized atom typeswill be optimized at the Hartree-Fock level of theory using a 6-31G(d)basis set. Upon convergence, partial charges of each atom will becomputed using Merz-Kollman charge fitting scheme (Besler et al. (1990)J. Computational Chemistry 11: 431-439). These charges can replace thedefault atomic charges.

2. Molecules to be simulated:

a. Construction of molecular structures

The molecules to be simulated can be built using QUANTA and the atomiccharges will be obtained as in step one above. The net charge of thewhole compound can then be computed.

b. Solvation of the molecular structures:

The molecules and molecular complexes constructed in step 2a can beneutralized using Na⁺ ions that are positioned using the SOLVATEprogram. The whole system can then be solvated in a box of watermolecules and this simulation box can be subsequently adjusted to yieldthe appropriate density.

B. Running the Simulation:

1. Equilibration of the molecule/water/counterion system:

a. Minimization: To remove residual strain remaining in the molecularstructures from the construction phase, the solvated molecules from step2b above can be minimized for 10,000 steps, of which the first 1,000iterations are performed using steepest descent and the rest usingadopted basis Newton-Raphson methods.

b. Equilibration: After minimization, The temperature can be ramped upfrom OK to 300K over 10 ps and held fixed at 300K thereafter. The systemcan be equilibrated for 200 ps at constant temperature. The long rangeforces are handled by particle mesh Ewald method Essmann et al. (1995)J. Chemical Physics 103: 8577-8593. The water molecules are TIP3P(Jorgensen et al. (1983) J. Chemical Physics 79: 926-935). Anintegration time step of 2 femtoseconds can be used, and the SHAKEalgorithm can be employed to restraint all the motions of the hydrogenatoms (Reichert and Welch (2001) Coordination Chemistry Reviews 212:111-131).

c. Production runs: For the production simulations, constant temperaturemolecular dynamics (using the NVT ensemble) can be used. The particlemesh Ewald method (Essmann et al. (1995) J. Chemical Physics 103:8577-8593) can be used for the long range forces. During the dynamicsruns, the complete set of atomic coordinates can be saved every 0.1 psfor subsequent analysis. For the preliminary simulations, the moleculardynamics simulations ran on our Compac Alpha computers at a speedcorresponding to approximately 625 cpu hours (˜4 weeks) per nanosecond,therefore, multi-nanosecond simulations of these systems will beroutinely feasible on our large network of workstations.

Example 3. Synthesis and Testing of a Bi-Denatate Shal

The bivalent SHAL (LeacPLD)₂LPDo was synthesized, purified by HPLC andcharacterized by mass spectrometry. This SHAL has two JP459B bidentateligands interconnected via a linker and a DOTA attached on the third arm(see, e.g., FIG. 14).

SHALs were designed around an orthogonally protected lysine residue tofacilitate synthesis on solid phase resin. A commercially availableFmoc-protected amino acid-like mini-peg (2 CH₂O's) was used as a linkerto incrementally increase the distance between the enkephalin and thedeoxycholate moieties. Fmoc-biotinyl-lysine was used to introduce biotininto the SHALs for biacore experiments. All SHALs follow the sameconfiguration: CO₂H:Biotin-lysine:lysine:(a lysine NH₂: 0, 1, 2 mini-peglinker, deoxycholate)(g lysine NH₂: LFGGY-NHAc). The bis-bidentate SHALfollows the convention: CO₂H:Biotin-lysine:lysine:[(a lysine NH₂: 0, 1,2 mini-peg linker, deoxycholate)(g lysine NH₂: LFGGY-NHAc)]2- andtherefore is unsymmetrical about the second lysine residue.

All chemicals used were purchased from Aldrich or Nova Biochem. SHALswere synthesized using standard Fmoc solid phase synthesis onchlorotritylchloride resin. Ligands were cleaved from the resin and theprotecting groups removed using the appropriate reagents.Trifluoroacetic acid esters formed on the primary alcohols ofdeoxycholate during cleavage from the resin were removed by stirring inammonium bicarbonate. SHALs were purified using reverse phase highperformance liquid chromatography (HPLC). Analytical HPLC was carriedout at 1 mL/min on an Agilent 1100 machine (Waters Symmetry C18, 5 mm,4.2×150 mm column) and preparative HPLC was carried out at 10 mL/min ona Waters preparative machine (Waters Symmetryprep C18, 7 mm, 19×300 mmcolumn). SHALs were characterized using nuclear magnetic resonance (NMR)spectroscopy and electrospray mass spectrometry. 1H and 13C NMR spectrawere recorded on a Bruker DRX 500 MHz spectrometer. Mass spectra wereacquired on a Micromass Quattro Micro API mass spectrometer operating inpositive ion mode.

Mass spectrometry of the (LeacPLD)₂LPDo showed that it does not containany free DOTA. Free DOTA would not be expected to be present based onthe process used to synthesize the SHAL. (LeacPLD)₂LPDo was synthesizedby attaching each linker component or ligand onto a growing chaincovalently attached to the surface of a resin. After each chemicalreaction the resin was extensively washed to remove the unreactedproducts. DOTA was attached to the linker at the beginning of thesynthesis. After the excess DOTA was washed away, multiple additionalchemical reactions that were carried out on the resin to add the variouslinkers and ligands, and after each reaction the unreacted products wereagain washed away. By the time the synthesis of the SHAL was completed,the amount of free DOTA present in the sample was undetectablyexamination of the HPLC and mass spectrum. The DOTA link is extremelystable, so it does not come off the SHAL once it's been attached.

During radiochemistry, components that may be present in the earlyrunoff peak that can have associated radioisotopic label include thebuffer salt (ammonium bicarbonate), possibly a trace amount oftrifluoroacetate removed from the SHAL hydroxyls during the finalsynthesis step, and excess EDTA and free and EDTA-complexed isotope thatdidn't bind to the DOTA. Dialysis may simply not be the preferred methodfor efficiently eliminating all this material. Reverse dialysis is onepreferred method for purification. Alternatively, chelate (EDTA)scrubbing after radiochemistry can be performed using an EDTA beadcolumn to remove radiometal that has not been DOTA chelated or isloosely attached in a non-specific manner.

To get the metal into the DOTA efficiently, adjusting the reaction mixto an adequate alkaline pH is also important. Since the SHAL as amolecule is quite different from an antibody-DOTA molecule, the methodused to raise the pH on the SHAL-DOTA complex preferably also raises thepH sufficiently on the SHAL. One can easily get other metals in DOTA ifthey are present at any stage. These can be detected by checking themass spectrum of the compound. We have looked at the spectrum frompurified SHALs and see little or no other metal there.

Biotinylated deoxycholate-iodothyronine SHALs are synthesized in ananalogous manner.

ELISA assays showed that SHAL, LeacPLBD (the univalent bidentate SHAL),bind to and discriminate between cells containing HLA-DR10 and thosethat do not contain HLA-DR10 (see FIG. 20).

Several pharmacokinetic, biodistribution and imaging studies wereperformed with consistent results. In certain embodiments, ¹¹¹In-DOTA[SHAL 070804(LeacPLD)₂LPDo] biodistribution was determined in Rajitumored mice (see, e.g., FIG. 19A).

Example 4. Development of Synthetic High Affinity Ligands that Bind to aStructural Epitope on the Tumor Cell Receptor HLA-DR10

A number of cell surface protein receptors have been identified thatdistinguish malignant cancer cells from their normal counterparts. Whileseveral of these receptors have been shown to be more abundant on cancercells (e.g., CD20, CD22), others have been confirmed to differstructurally (e.g., HLA-DR10, Muc-1) from the same or closely relatedreceptors present on normal cells. This has led to the development of avariety of antibody-based, receptor-specific reagents conjugated toradioisotopes for use in radio-immunotherapy. Lym-1, an antibodydeveloped to bind to a unique structural epitope on the abundant cellsurface receptor HLA-DR10 found only on human lymphoma and normal B-celllymphocytes, has been used with some success in the treatment of nonHodgkin's lymphoma. In an effort to develop smaller and more effectivetherapeutics for treating non Hodgkin's lymphoma, we have synthesizedthe first in a series of small (<3 kD) selective high affinity ligands(SHALs) that bind to this same HLA-DR10 structural epitope. A homologymodel for HLA-DR10 was created using four known crystal structures ofrelated HLA-DR molecules. Two unique “pockets” located on the surface ofthe protein adjacent to key amino acids required for Lym-1 binding wereidentified, and computational docking techniques were used to prescreena large library of small molecules to predict which compounds shouldbind to each site. A small number of these compounds were testedexperimentally using NMR spectroscopy to confirm their binding to theisolated HLA-DR10 protein, and pairs of compounds binding to the twodifferent “pockets” were linked together using solid phase syntheticchemistry to create a series of bidentate reagents with different lengthlinkers. The SHAL exhibiting the highest affinity for isolated HLA-DR10(˜23 nM) has been shown to bind selectively to nine different culturedcell lines containing HLA-DR10 and to frozen and fixed tissue sectionsobtained from patients with small cell and large cell human lymphomas. Abivalent form of this SHAL was also synthesized and shown to furtherenhance cell binding.

Example 5. Hexa-Arginine Enhanced Uptake and Residualization ofSelective High Affinity Ligands by Raji Lymphoma Cells Summary

A variety of arginine-rich peptide sequences similar to those found inviral proteins have been conjugated to other molecules to facilitatetheir transport into the cytoplasm and nucleus of targeted cells. Theselective high affinity ligand (SHAL) (DvLPBaPPP)₂LLDo, which wasdeveloped to bind only to cells expressing HLA-DR10, has been conjugatedto one of these peptide transduction domains, hexa-arginine, to assessthe impact of the peptide on SHAL uptake and internalization by Rajicells, a B-cell lymphoma.

An analog of the SHAL (DvLPBaPPP)₂LLDo containing a hexa-argininepeptide was created by adding six D-arginine residues sequentially to alysine inserted in the SHAL's linker. SHAL binding, internalization andresidualization by Raji cells expressing HLA-DR10 were examined usingwhole cell binding assays and confocal microscopy. Raji cells wereobserved to bind two fold more ¹¹¹In-labeled hexa-arginine SHAL analogthan Raji cells treated with the parent SHAL. Three fold morehexa-arginine SHAL remained associated with the Raji cells afterwashing, suggesting that the peptide also enhanced residualization ofthe ¹¹¹In transported into cells. Confocal microscopy showed both SHALslocalized in the cytoplasm of Raji cells, whereas a fraction of thehexa-arginine SHAL localized in the nucleus.

The incorporation of a hexa-D-arginine peptide into the linker of theSHAL (DvLPBaPPP)₂LLDo enhanced both the uptake and residualization ofthe SHAL analog by Raji cells. In contrast to the abundant cell surfacebinding observed with Lym-1 antibody, the majority of(DvLPBaPPP)₂LArg6AcLLDo and the parent SHAL were internalized. Some ofthe internalized hexa-arginine SHAL analog was also associated with thenucleus. These results demonstrate that several important SHALproperties, including uptake, internalization, retention and possiblyintracellular distribution, can be enhanced or modified by conjugatingthe SHALs to a short polypeptide.

Background

Several strategies have been used to selectively deliver toxic chemicalsor radiation to cancer cells (DeNardo (2005) Semin Oncol., 32:S27-35;Torchilin (2007) AAPS J., 9:E128-147), for gene therapy (Jeong et al.(2005) J Control Release 107:562-570; Xia et al. (2007) J Gene Med10:306-315) or as tools for transfecting cells (Shigeta K (2007) JControl Release 118:262-270) and silencing genes (Liu (2007) Brief FunctGenomic Proteomic 6:112-119). Some of the earliest approaches used toenhance the cellular uptake of therapeutics and other molecules(fluorescent dyes, enzymes, antibodies and other proteins) involvedintroducing the molecules into liposomes or micelles (Constantinides etal. (2008) Adv Drug Deliv Rev 60:757-767; Samad et al. (2007) Curr DrugDeliv 4:297-305). Such constructs have been shown to fuse with thecell's membrane, introducing the contents inside the cell ortransferring the lipid-bound components into the cell's membrane.Another highly successful approach has been to develop antibodies thattarget cell-specific membrane proteins and to use these antibodies todeliver radionuclides or other cytotoxic molecules to the surface of aspecific population of cells (Brumlik et al. (2008) Expert Opin DrugDeliv 5:87-103; DeNardo et al. (1998) Cancer Biother Radiopharm13:239-254; Tolmachev et al. (2007) Cancer Res. 67:2773-2782). Morerecently, intracellular delivery has been accomplished by attaching themolecules to be transported to naturally occurring transmembrane“shuttles”, peptides or proteins that readily pass through cellularmembranes. One of the more successful shuttles is a nuclear localizationsignal peptide derived from the SV40 T antigen (Yoneda (1997) J Biochem121:811-817). This sequence, other peptide sequences derived from thetransduction domain of the HIV-1 protein Tat (Schwarze et al. (1999)Science 285:1569-1572; Torchilin et al. (2003) Proc. Natl. Acad. Sci.,USA, 100:1972-1977), penetratin (Tseng et al. (2002) Mol Pharmacol62:864-872), and intact proteins such as the herpes virus protein VP22(Phelan et al. (1998) Nat Biotechnol 16:440-443) and anti-DNA antibodies(Avrameas et al. (1998) Proc. Natl. Acad. Sci., USA, 95:5601-5606) arecurrently being used to facilitate the transport of liposomes, viruses,enzymes, antibodies and a variety of other proteins into cells.Considerable success has also been achieved using synthetic cationicpeptide transporters such as oligoarginine (Futaki (2005) Adv Drug DelivRev 57:547-558; Han et al. (2001) Mol Cells 12:267-271; Kim et al.(2007) Int J Pharm 335:70-78; Tung and Weissleder (2003) Adv Drug DelivRev 55:281-294), lactosylated poly-L-lysine (Midoux et al. (1993)Nucleic Acids Res 21:871-878) and short peptide sequences selected fromphage display libraries (Kamada et al. (2007) Biol Pharm Bull 2007,30:218-223) that exhibit sequence similarities to know peptide shuttles.

Recently, several small molecule antibody mimics that show promise astargeting agents for cancer imaging or therapy have been synthesized[24-28]. In addition to exhibiting selectivities and affinities (nM topM) similar to antibodies, these molecules have the potential tominimize some of the difficulties associated with the use ofprotein-based drug delivery systems. They retain the more desirablepharmacokinetic properties of small molecules, are less likely to beimmunogenic, may prove stable enough for oral delivery, and the costsassociated with producing the drug can be reduced significantly. TheSHAL family of antibody mimics can also be easily modified to carryradioactive metals, a variety of tags that enable their use as imagingagents, and other small molecules (e.g. toxins or inhibitors). Anotherpotentially useful modification includes alterations that facilitateuptake and internalization of the SHAL by the targeted cell, which wouldbe expected to both increase tumor residence time and deliver the SHALinto an environment (the cytoplasm or nucleus) where it could causeadditional damage.

Working with a SHAL developed previously for targeting HLA-DR10, anabundant cell surface receptor over-expressed on B-cell malignancies, wesynthesized a peptide analog to the SHAL by conjugating it tohexa-arginine, a peptide that has been demonstrated previously tofacilitate the transport of proteins and nucleic acids into cells.Binding studies conducted with the SHAL and its hexa-arginine analog invitro using HLA-DR10 expressing Raji cells show that the hexa-argininesequence changed the SHALs properties significantly, enhancing both SHALinternalization and radionuclide residualization.

Methods

SHAL Design.

The process used to create a homology model for HLA-DR10, identifyunique binding cavities within the Lym-1 epitope, select ligands thatbind in these cavities, and create the (DvLPBaPPP)₂LLDo SHAL has beenreported previously (Balhorn et al. (2007) Clin Cancer Res13:5621s-5628s). A process for producing a hexa-arginine peptide analogof this parent SHAL, (DvLPBaPPP)₂LArg₆AcLLDo, was developed by modifyingthe synthesis to include the incorporation of an additional lysineresidue into the middle of the linker connecting the two SHAL monomersand attaching an arginine hexapeptide to the free amine on this lysine.

SHAL Synthesis.

The two dimeric SHALs (DvLPBaPPP)₂LLA and (DvLPBaPPP)₂LArg₆AcLLA weresynthesized on chlorotrityl chloride resin using orthogonally protectedlysine (L) residues and miniPEGs (P) to link the two small ligands Dvand Ba as previously described for (DvLPBaPPP)₂LLA (Balhorn et al.(2007) Clin Cancer Res 13:5621s-5628s; Hok et al. (2007) Bioconjug Chem18:912-921). To produce the amine derivative of the hexa-arginine SHAL(DvLPBaPPP)₂LArg₆AcLLA, a second Dde-D-Lys(Fmoc)-OH lysine residue wasinserted into the linker during SHAL synthesis by performing twosequential Dde-D-Lys(Fmoc)-OH coupling steps. At the alpha position ofthe third lysine, six consecutive arginine residues were inserted byreacting the resin with Fmoc-D-Arg(Pbf)-OH six times. The sixth Argresidue was protected with an acetate (Ac) by reacting with aceticanhydride in N,N diisopropyl-ethylamine (DIEA)/dimethylformamide (DMF).The guanidinium groups on all six arginine residues remain protectedwith trifluoroacetic acid (TFA)-sensitive2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) protectinggroups throughout the rest of the synthesis. The remainder of thesynthesis was then completed as described previously for (DvLPBaPPP)₂LLA(Id.). Analytical HPLC and electrospray ionization mass spectrometry(ESI-MS) were performed to confirm the purity and identity of the(DvLPBaPPP)₂LLA and (DvLPBaPPP)₂LArg₆AcLLA free amine SHALs.

(DvLPBaPPP)₂LLA:

Starting with 50 mg (0.07 mmol) resin and 30 mg (0.07 mmol)Fmoc-D-Lys(Boc)-OH, 34 mg of (DvLPBaPPP)₂LLA (Rt=7.86 min, WatersSymmetry C18, 5 μm, 4.2×150 mm column, diode array detector with alinear gradient from 95% H₂O, 1% TFA to 80% acetonitrile (MeCN), 1% TFAover 12 min) was isolated as red solid after purification. ESI-MS: m/zcalculated for C₁₅₀H₂₂₄N₃₄O₄₁S₂ (M+ 3H)³⁺ 1075.60, found 1075.62;calculated for (M+ 4H)⁴⁺ 806.95, found 806.93; calculated for (M+ 5H)⁵⁺645.76, found 645.68; calculated for (M+6H)⁶⁺ 538.30, found 538.21.

(DvLPBaPPP)₂LArg₆AcLLA:

81 mg of (DvLPBaPPP)₂LArg₆AcLLA (Rt=8.30 min) starting from 90 mg (0.12mmol) resin and 154 mg (0.29 mmol) Fmoc-D-Lys(Boc)-OH was isolated asred solid after purification. ESI-MS: m/z calculated forC₁₉₄H₃₁₀N₆₀O₄₉S₂ (M+ 3H)³⁺ 1444.71, found 1444.65; calculated for (M+4H)⁴⁺ 1083.76, found 1083.78; calculated for (M+ 5H)⁵⁺ 867.23, found867.18; calculated for (M+ 6H)⁶⁺ 722.86, found 722.78; calculated for(M+7H)⁷⁺ 619.74, found 619.62.

Attachment of DOTA to SHALs.

The amine analog of the SHAL (DOTA-SHAL precursor with a free epsilonamine on the first lysine) was dissolved in 500 μl anhydrous DMF and 100μl DIEA. The hexafluorophosphate (PF₆) salt of DOTA N-hydroxysuccinimide(NHS) ester (933.36 g/mol, 1-1.5 equivalents) was added to the mixtureas a solid. The mixture was nutated for 15 min and the reaction wasmonitored by analytical HPLC. Upon completion the reaction solution wasdiluted with 300 μl H₂O and 300 μl MeCN (both containing 1% TFA) andHPLC purified using an 85% H₂O (0.1% TFA) to 70% MeCN (0.1% TFA)gradient run over 25 min. The resulting purified DOTA-SHALs werelyophilized and subsequently analyzed by analytical HPLC (WatersSymmetry C18, 5 μm, 4.2×150 mm column, diode array detector) using alinear gradient from 95% H₂O (1% TFA) to 80% MeCN (1% TFA) over 12 min)and characterized by ESI-MS.

(DvLPBaPPP)₂LLDo:

Reaction of the (DvLPBaPPP)₂LLA amine SHAL (6.0 mg, 1.86 μmol) with DOTANHS ester (2.0 mg, 2.14 μmol) gave 100% (Rt=7.664 min) conversion bycrude analytical HPLC and yielded (DvLPBaPPP)₂LLDo (8.0 mg, red solid)after purification. ESI-MS: m/z calculated for C₁₆₆H₂₅₀N₃₈O₄₈S₂ (M+2H)²⁺1806.09, found 1806.22; calculated for (M+3H)³⁺ 1204.40, found 1204.49;calculated for (M+4H)⁴⁺ 903.55, found 903.61; calculated for (M+5H)⁵⁺723.04, found 723.07; calculated for (M+ 6H)⁶⁺ 602.70, found 602.64.

(DvLPBaPPP)₂LArg₆AcLLDo:

Reaction of (DvLPBaPPP)₂LArg₆AcLLA amine SHAL (15.0 mg, 3.46 μmol) withDOTA NHS ester (5.0 mg, 5.36 μmol) gave 100% (Rt=7.70 min) conversion bycrude analytical HPLC and yielded (DvLPBaPPP)₂LArg₆AcLLDo (12.0 mg, redsolid) after purification. ESI-MS: m/z calculated for C₂₁₀H₃₃₆N₆₄O₅₆S₂(M 3H)³⁺ 1573.51, found 1573.54; calculated for (M+ 4H)⁴⁺ 1180.38, found1180.43; calculated for (M+ 5H)⁵⁺ 944.51, found 944.52; calculated for(M+ 6H)⁶⁺ 787.26, found 787.26; calculated for (M+7H)⁷⁺ 674.94, found674.88; calculated for (M+ 8H)⁸⁺ 590.69, found 590.58.

Radiochemistry.

As described previously (Id.), the DOTA-SHALs were labeled withcarrier-free ¹¹¹InCl₃ (MDS Nordion, Vancouver, Canada) using thefollowing method (DeNardo et al. (2007) J Nucl Med 48:1338-1347). Analiquot of ¹¹¹InCl₃ (15-20 μl) was added to a solution of DOTA-SHAL(25-50 μg) in 0.1 M NH₄OAc, pH 5.3 (50 μl); the final pH of the reactionmixture was adjusted to 6.5 by adding 4M NH₄OAc and the mixture wasincubated for 1 h at 37° C., then 10-20 μl of 0.1 Methylenediaminetetraacetic acid (EDTA) was added to sequester excess,free ¹¹¹In³⁺. The radiolabeled product was purified using HPLC, followedby dialysis in phosphate-buffered saline (PBS) with a 1 kD cut offmembrane. The purity of the ¹¹¹In-labeled SHALs was determined by thinlayer chromatography (TLC) (10% NH₄OAc-MeOH 1:1), HPLC and celluloseacetate electrophoresis (CAE). CAE resolved ¹¹¹In-DOTA-SHALs and¹¹¹In-EDTA; radioactive peaks were observed at 2.3-3.0 cm and >6.5 cm,respectively. Similar results were observed in the TLC assay;¹¹¹In-DOTA-SHALs showed little migration from the point of application(R_(F)=0.25-0.3), whereas ¹¹¹In-EDTA moved towards the solvent front(R_(F)=0.5). By HPLC, ¹¹¹In-EDTA eluted at 2.5-3.0 ml and¹¹¹In-DOTA-SHALs at 9.5-10 ml. The ¹¹¹In labeled SHALs were purifiedusing RP-HPLC or a 1 kD dialysis membrane in PBS, and concentrated usinga Savant Speedvac SC110 (Thermo Fisher Scientific, Inc, Waltham, Mass.,USA). Final radiochemical purity was determined using C18-RP-TLC (EMScience, DC-Plastikfolien kieselgel 60 F254, Cherry Hill, N.J.), HPLC,and CAE. ¹¹¹In-DOTA-SHAL product yields ranged from 70-90% and thepurity of the product ranged from 90-95%. The final product wasdissolved in 10% dimethylsulfoxide (DMSO) in PBS, and proved stable over72 hours at room temperature.

SHAL Binding to Isolated HLA-DR10 Protein

Protein binding experiments were conducted using surface plasmaresonance on a Biacore 3000 (Biacore, Piscataway, N.J.) at 25° C. Aresearch grade streptavidin immobilized chip (SA chip, Biacore) waspreconditioned and normalized according to the manufacturersinstructions. Biotin labeled SHALs were dissolved in DMSO and diluted in1.05×PBS (Biacore) to a final concentration of 1×PBS pH 7.4, 5% DMSO, tomatch the running buffer. These SHALs were injected over the flow cellto yield a surface density of 500-1000 RU (response units). Biotin (50μM E-Z Link Amine-PEO2-Biotin, Pierce) was injected over all cells for 1minute at 20 μl/min as a block to reduce non-specific binding. One flowcell was used as a reference cell and a different SHAL was immobilizedon each of the three other cells.

Experiments measuring the binding of HLA-DR10 to the SHALs were carriedout at a flow rate of 30 μl/minute in PBS pH7.4 running buffer using all4 flow cells. HLA-DR10 isolated from Raji cells (Rose et al. (1996)Cancer Immunol Immunother 43:26-30) was diluted in running buffer to afinal concentration ranging from 10 nM to 1 μM, and a series ofconcentrations were run randomly in triplicate. Protein was injected for3 minutes, allowed to dissociate for 5 minutes followed by regenerationof the surface using a 1 minute injection of 0.1% sodium dodecylsulfate(SDS) followed by a washing step with a 2 minute injection of runningbuffer. The data, which were double referenced by subtracting the blankreference surface and an average of 5 blank injections, were processedusing the program SCRUBBER (University of Utah).

Cell Binding Assay

Raji human Burkitt's lymphoma B-cells (American Type Culture Collection,Manassas, Va.) were maintained in RPMI-1640 media supplemented with 10%fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% of asolution of nonessential amino acids (GIBCO #11140-050), and 100units/ml of Penicillin G, 100 μg/ml Streptomycin, and 0.25 μg/ml ofAmphotericin B at 37° C. in a humidified 5% CO₂ atmosphere. Jurkat'scells (American Type Culture Collection, Manassas, Va.), an acuteleukemia T-cell line, were maintained in the same medium with theaddition of 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES).

A series of experiments were conducted to quantify the uptake of the¹¹¹In-labeled parent SHAL (DvLPBaPPP)₂LLDo and its hexa-arginine analog(DvLPBaPPP)₂LArg₆AcLLDo by Raji cells, a cell line that has beenpreviously shown to express the HLA-DR10 variant. The assays wereconducted using aliquots containing 10⁶ cells suspended in 150 μl of PBSwith 5% bovine serum albumin (BSA). Aliquots of cells were treated with0.1, 1, 5, 10 or 25 ng of ¹¹¹In-labeled (DvLPBaPPP)₂LLDo or(DvLPBaPPP)₂LArg₆AcLLDo for one hour at both 4° C. and 22° C. The tubescontaining the treated cells were centrifuged to separate the cellpellet from the supernatant and the two fractions were counted in acalibrated gamma well counter to determine the amount of bound and freeSHAL. Half of the cell pellets were washed twice with PBS and incubatedat 22° C. for 15 min before centrifuging them again. The pooled washesand washed cell pellets were subsequently counted in the gamma wellcounter to assess how much of the bound SHAL could be removed bywashing.

3-D Confocal Microscopy

SHAL binding and internalization by Raji and Jurkat's cells was assessedusing the method described previously by O'Donnell et al. (O'Donnell etal. (1998) Prostate 37:91-97). Experiments were conducted comparing thebinding of (DvLPBaPPP)₂LLDo (the parent SHAL), its hexa-arginine analog(DvLPBaPPP)₂LArg₆AcLLDo, and chimeric Lym-1 (chLym-1) to Raji cells. Allsteps were performed at 20° C., unless indicated.

Four million Raji cells (>92% viability) in log phase growth werepelleted at 300×g, washed, and blocked for 30 min in 1 ml of 1% fractionV BSA in PBS, with constant rotation. Cells were then incubated 1 hr, at1 million per 250 μl, with either 1% BSA in PBS or a biotinylatedprimary reagent: 10 nM chLym-1, 10 μM parent SHAL, or 10 μMhexa-arginine SHAL. After four washes (two in 1% BSA in PBS, two inPBS), 50 μl of the cell suspensions was applied to freshly poly-L-Lysinecoated slides, and cells were allowed to adhere for 10 min in a humidchamber. Fixation and permeabilization were performed at −20° C. byusing a 4 min exposure to methanol. Jurkat's cells were treated in thesame manner as a control.

Slides were then washed twice in PBS and blocked in 10% fetaplex serum(Gemini Bioproducts, West Sacramento, Calif.) in PBS for 15 min andwashed once in PBS. The detection reagent, Streptavidin AlexaFluor 610(Invitrogen, Carlsbad, Calif.) was diluted 1/500 in diluent, 100 μl wasapplied; a parafilm cover slip was layered over the solution to preventevaporation. The slides were incubated in a humid chamber for 30 min.,washed 5 times for 5 min each in PBS, and rinsed briefly in doubledistilled H2O. After the slides dried, cover slips were mounted withProlongGold with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen,Carlsbad, Calif.). The slides were viewed with an Olympus FV1000 laserscanning confocal microscope and data were collected as Z-scans at 160×,with focal sections being taken 1 μm apart through the cell.

Statistical Analysis

Data is reported as mean±SD. Statistical comparisons were based on theWilcoxon rank sum test (Hollander and Wolfe (1973) Nonparametricstatistical methods. New York: Wiley Publications), a procedure based onranking the values of two test groups. Differences were consideredstatistically significant if p values were ≦0.05. The p-values weredetermined by the transformation Z=TANH⁻¹r for the correlationcoefficients (CRC Handbook of Tables for Probabilities and Statistics.2nd edn. Boca Raton, Fla.: CRC Press; 1968).

Results

SHAL Design and Synthesis.

Two forms of the free amine SHAL, (DvLPBaPPP)₂LLA, and the hexa-arginineanalog, (DvLPBaPPP)₂LArg₆AcLLA, were synthesized in multi-milligramamounts and purified by high performance liquid chromatography (HPLC). Abiotin was attached to the ε-amino group of the terminal amine (A) onboth (DvLPBaPPP)₂LLA and (DvLPBaPPP)₂LArg₆AcLLA to produce biotinylatedforms for use in cell and protein binding experiments.1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) wasattached to both (DvLPBaPPP)₂LLA and (DvLPBaPPP)₂LArg₆AcLLA at the samesite to enable the SHALs to be labeled with ¹¹¹In. The DOTA SHAL(DvLPBaPPP)₂LLDo (FIG. 25A) and the hexa-arginine SHAL analog(DvLPBaPPP)₂LArg₆AcLLDo (FIG. 25B) were labeled with ¹¹¹In at highefficiency (>90%) with specific activities ranging from 70-85 μCi/μgSHAL. Analyses of the resulting radiolabeled SHAL by HPLC and celluloseacetate electrophoresis (CAE) showed the purity of the product to begreater than 90%. D-isomers of arginine incorporated during thesynthesis of the hexa-arginine sequence in (DvLPBaPPP)₂LArg₆AcLLDo wereused to minimize the proteolytic susceptibility of the peptide. Whilemore detailed experiments need to be carried out to adequately assessthe stability of the SHAL in vivo, data obtained from one preliminaryCAE experiment showed no evidence of degradation when thehexa-D-arginine SHAL analog was incubated in human plasma at 37° C. for24 hrs (data not shown).

SHAL Affinity for HLA-DR10 Protein.

Surface Plasmon resonance binding studies were conducted with both SHALsto estimate and compare the affinity of the two SHALs for isolatedHLA-DR10 protein. In a series of kinetic experiments in whichbiotinylated versions of the SHALs were immobilized on the surface of astreptavidin chip, the parent SHAL (DvLPBaPPP)₂LLDo was observed to bindto HLA-DR10 with a Kd˜21 nM. A similar Kd, ˜34 nM, was obtained for thehexa-arginine containing analog (DvLPBaPPP)₂LArg₆A cLLDo.

Analysis of SHAL Uptake by Raji Cells Expressing HLA-DR10.

In vitro cell binding experiments were conducted using ¹¹¹In-labeledparent SHAL and the hexa-arginine SHAL analog to quantify SHAL uptakeand to evaluate the effect of adding the hexa-arginine tag. Uptake wasassessed using Raji cells, a lymphoma cell line expressing HLA-DR10.Aliquots containing 10⁶ cells were incubated with increasing amounts ofSHAL containing ¹¹¹In labeled SHAL as a tracer, and cell-associated¹¹¹In was measured before and after washing the cell pellets.

Analyses of the unwashed cell pellets showed that both the parent SHALand the hexa-arginine SHAL are bound by Raji cells. Cell associated SHALincreased linearly with increasing SHAL concentration in the media forboth SHALs (FIG. 26), and the amount of bound SHAL showed no evidence ofreaching saturation over the range of SHAL concentration tested. Rajicells treated with the hexa-arginine SHAL, in contrast to those treatedwith the parent SHAL, bound twice as much SHAL (Table 9). A largerproportion of the hexa-arginine SHAL (67%) was also retained by thecells after washing when compared to the parent SHAL (˜46%), leading toa final hexa-arginine SHAL content three times that of its parent.

TABLE 9 Retention (residualization) of bound SHAL by Raji cells.Identical samples of cells used in experiments shown in FIG. 27 wereincubated as described in above with 5.3 pmoles of ₁₁₁In-labeled(DvLPBaPPP)₂LLDo or (DvLPBaPPP)₂LArg₆AcLLDo and then washed two timeswith BSA/buffer. The cell pellets were then counted to provide estimatesof SHAL remaining bound after washing. Percent SHAL pmoles SHALBound/10⁶ cells Re- SHAL unwashed Washed tained (DvLPBaPPP)₂LLDo 0.568 ±0.091 0.263 ± 0.000 46 (DvLPBaPPP)₂LArg₆AcLLDo 1.300 ± 0.038 0.876 ±0.017 67

SHAL Localization by 3-D Confocal Microscopy.

Fluorescence images collected at focal planes near the center of Rajicells treated with biotinylated forms of the parent and hexa-arginineSHALs for only an hour confirmed that both SHALs were taken up by Rajicells (FIG. 27). In contrast to Lym-1 antibody, which binds to HLA-DR10on the cell surface, the sectioned images taken from the center of thecells showed that both SHALs were localized inside Raji cells anddistributed throughout the cytoplasm. Raji cells took up significantlymore of the hexa-arginine SHAL than the parent SHAL, as evidenced by themore intense staining of the cytoplasm of cells treated with equivalentconcentrations of the two SHALs. SHAL uptake was not observed in controlJurkat's cells (cells lacking HLA-DR10). A fraction of the hexa-arginineSHAL also appeared to be associated with the nucleus. Nuclear stainingwas not observed in cells treated with the parent SHAL.

Discussion

Numerous cell penetrating peptides (CPPs) derived from viral and otherproteins that traverse cell and nuclear membranes have been employed asshuttles to improve the efficiency of transport of liposomes, exogenousproteins and nucleic acids, and other molecules into the cytoplasm andnuclei of cells (Schwarze et al. (1999) Science 285:1569-1572; Torchilinet al. (2003) Proc. Natl. Acad. Sci., USA, 100:1972-1977; Tseng et al.(2002) Mol Pharmacol 62:864-872; Phelan et al. (1998) Nat Biotechnol16:440-443; Avrameas et al. (1998) Proc. Natl. Acad. Sci., USA,95:5601-5606; Futaki (2005) Adv Drug Deliv Rev 57:547-558; Han et al.(2001) Mol Cells 12:267-271; Kim et al. (2007) Int Pharm 335:70-78; Tungand Weissleder (2003) Adv Drug Deliv Rev 55:281-294; Midoux et al.(1993) Nucleic Acids Res 21:871-878; Kamada et al. (2007) Biol PharmBull 2007, 30:218-223). Studies characterizing the efficiency ofinternalization of different CPP sequences, all of which have a highcontent of arginine residues (Futaki (2006) Biopolymers 84:241-249),have shown that arginine homopolymers containing as few as six arginineresidues are highly effective in transporting small organic molecules(Kirschberg et al. (2003) Org Lett 5:3459-3462; Rothbard et al. (2000)Nat Med 6:1253-1257) and large proteins into cells (Futaki et al. (2001)J Biol Chem 276:5836-5840).

In an effort to develop SHALs that are more efficiently internalized andresidualized by the cells they target, we synthesized a hexa-arginineconjugate of (DvLPBaPPP)₂LLDo, a SHAL containing the two ligandsdabsylvaline (Dv) and N-benzoyl-L-arginyl-4-amino benzoic acid (Ba) thathad been shown previously to bind selectively to HLA-DR10 expressingcell lines (Balhorn et al. (2007) Clin Cancer Res 13:5621s-5628s).Hexa-arginine was chosen as the first shuttle sequence to be tested forits ability to facilitate the transport of SHALs into cells because itcould be conjugated to a dimeric SHAL without changing its molecularmass significantly, thereby preserving the desirable properties of theSHAL as a small molecule therapeutic. Surface Plasmon resonanceexperiments comparing the binding of the SHAL and the hexa-arginine SHALanalog to purified HLA-DR10 protein showed that the addition of thehexa-arginine peptide to the dimeric SHAL did not interfere with SHALbinding to the protein.

3-D Confocal microscopy experiments revealed that both the parent SHALand its hexa-arginine analog were taken up and internalized by HLA-DR10expressing Raji cells. SHAL uptake was not observed in Jurkat's cells, acell line lacking HLA-DR10. Optical sections taken through Raji cellsshowed that the binding of the SHALs was not confined to the cellsurface, as is characteristic of Lym-1 antibody binding. Mid-planesections taken from cells treated with the SHALs showed theSHAL-associated fluorescence to be distributed throughout the interiorof the cells. In some images, areas of high SHAL concentration withinthe cytoplasm occasionally appeared to be associated with smallorganelle-like structures. The cytoplasm-associated fluorescence wassignificantly higher in Raji cells treated with the hexa-arginine SHALanalog, suggesting the addition of the hexa-arginine peptide enhancedcell uptake of the SHAL.

Experiments comparing the binding of ¹¹¹In-labeled (DvLPBaPPP)₂LLDo and(DvLPBaPPP)₂LArg₆AcLLDo to live Raji cells confirmed that thehexa-arginine tag enhanced SHAL uptake. The presence of the tag alsoincreased the amount of ¹¹¹In-labeled SHAL that was retained by Rajicells. The amount of SHAL retained after washing did not reachsaturation over the concentration range tested, suggesting that evenhigher concentrations of SHAL may be accumulated inside HLA-DR10expressing cells than achieved in these experiments. At the highestconcentration of hexa-arginine SHAL tested in the cell binding studies,the amount of residualized SHAL was equivalent to ˜1.1×10⁶ SHALmolecules per cell—the same number of HLA-DR10 molecules reportedpreviously to be present on the surface of each Raji cell (Epstein etal. (1987) Cancer Res 47:830-840). These results, together with theconfocal images showing the majority of the hexa-arginine SHAL isinternalized, indicate that a significant fraction of the SHAL may bebound to the pool of HLA-DR10 known to be present inside the cell.

The observed enhancement in residualization of ¹¹¹In-labeledhexa-arginine SHAL by Raji cells and the potential association of afraction of the ¹¹¹In-label with the nucleus are also important becauseradioisotope internalization and residualization have been shown to behighly advantageous for cancer therapy (Brouwers et al. (2003) ClinCancer Res 9:3953S-3960S; Chen et al. (2006) J Nucl Med 2006,47:827-836; Michel et al. (2002) Clin Cancer Res 8:2632-2639; Stein etal. (2995) Clin Cancer Res 11:2727-2734). Cancer therapeutics have beenlinked to a variety of radioisotopes that emit beta particles, alphaparticles or Auger electrons. The range of beta emissions from isotopesroutinely used in radioimmunotherapy, such as iodine-131, yttrium-90,and rhenium-188, extend for several millimeters, and therapeuticscarrying these radionuclides create a “crossfire” (DeNardo (2005) SeminOncol., 32:S27-35; Bischof 92003) Leuk Lymphoma 44 Suppl 4:S29-36) or“bystander” (Boyd et al. (2006) J Nucl Med 47:1007-1015) effectdestroying malignant cells to which the targeting agent is not directlybound. In this way, beta-emitters can potentially overcome resistancedue to antigen-negative tumor cells. These characteristics makebeta-particle therapy better suited for treating bulky tumors orlarge-volume disease. However, longer-ranged beta emissions can alsodestroy nearby normal cells.

The internalization of targeting agents such as the hexa-arginine SHAL(DvLPBaPPP)₂LArg₆AcLLDo can be exploited as a means of introducing Augerelectron-emitting ¹¹¹In into the cytoplasm and nucleus of cells wherethe Auger electrons have a very short, subcellular path length and highlinear energy transfer (Bodei et al. (2003) Cancer Biother Radiopharm18:861-877; Kassis (2003) J Nucl Med 44:1479-1481; McDevitt et al.(1998) Eur J Nucl Med 25:1341-1351). The radiation absorbed dose to thenucleus has been estimated to be 2-fold and 35-fold greater when ¹¹¹Indecays in the nucleus compared to when decay occurs in the cytoplasm oron the cell surface, respectively (Goddu et al. (1994) J Nucl Med35:303-316; Hindorf et al. (2007) Cancer Biother Radiopharm 22:357-366).These properties render ¹¹¹In and other Auger electron-emitters highlycytotoxic and damaging to DNA when they decay in close proximity to thecell nucleus (Costantini et al. (2007) J Nucl Med 48:1357-1368). Bycoupling Auger emitters to highly selective, residualizing targetingagents that accumulate to high concentrations inside tumor cells, a verypowerful class of therapeutics may be developed that are more effectivein treating many types of metastatic cancer.

Conclusions

The enhancement in hexa-arginine SHAL internalization by HLA-DR10expressing lymphoma cells and the magnitude of the increase in SHALresidualization achieved by conjugating a hexa-arginine peptide to theSHAL are important because they show that small molecules such as SHALscan be designed to deliver radionuclides to malignant cells underconditions that lead to residualization of significant concentrations ofradionuclide inside the cell. SHALs carrying Auger-emittingradionuclides may provide an alternative approach for increasing thetherapeutic index achieved with SHALs beyond that attained by theaccumulation of radionuclide-tagged targeting agents on the surface ofthe tumor cell. These results are also exciting because of the relevanceof the SHAL-based approach to treating other forms of cancer.Internalizing SHALs targeting under-glycosylated MUC1, the androgenreceptor and other tumor specific cell surface proteins that residualizethe radioisotopes they carry could also be developed as small moleculetherapeutics for a wide variety of other types of metastatic cancer.

Abbreviations Used in this Example.

Ac, acetate; Ba, N-benzoyl-L-arginyl-4-amino benzoic acid; Boc, tertiarybutyloxycarbonyl; BSA, bovine serum albumin; CAE, cellulose acetateelectrophoresis; CPP, cell penetrating peptide; DAPI,4′,6-diamidino-2-phenylindole; Dde,1-(4,4-dimethyl-2,6-dixoxcyclohex-1-ylidene)ethyl; DIEA, N,NDiisopropyl-ethylamine; DMF, dimethylformamide; DMSO, dimethylsulfoxide;DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; Dv,dabsylvaline; EDTA, ethylenediaminetetraacetic acid; ESI-MS,electrospray ionization mass spectrometry; Fmoc, fluorenylmethyloxy;HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HPLC, highperformance liquid chromatography; MeCN, acetonitrile; NHS,N-hydroxysuccinimide; Pbf,2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl; PBS, phosphatebuffered saline; PF6, hexafluorophosphate; RP-HPLC, reversed phase highperformance liquid chromatography; SDS, sodium dodecylsulfate; SHAL,selective high affinity ligand; TFA, trifluoroacetic acid; TLC, thinlayer chromatography.

Example 6. Molecular Specific and Cell Selective Cytotoxicity Induced bya Novel Synthetic HLA-DR Antibody Mimic for Lymphoma and LeukemiaSummary

Like rituximab, monoclonal antibodies reactive with human leukocyteantigen have potent antilymphoma activity. However, size limits theirvascular and tissue penetration. To mimic monoclonal antibody binding,nanomolecules have been synthesized, shown specific for the β subunit ofHLA-DR10, and selective for cells expressing this protein. Selectivehigh affinity ligands (SHALs) containing the3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionicacid (Ct) ligand residualized and had antilymphoma activity againstexpressing cells. Herein, we show the extraordinary potency in mice withhuman lymphoma xenografts of a tridentate SHAL containing this ligand.After titrating antilymphoma activity in cell culture, a randomizedpreclinical study of a tridentate SHAL containing the Ct ligand wasconducted in mice with established and aggressive human lymphomaxenografts. Mice having HLA-DR10 expressing Raji B- or Jurkat'sT-lymphoma xenografts were randomly assigned to receive either treatmentwith SHAL at a dose of 100 ng i.p. weekly for 3 consecutive weeks, or tobe untreated. Primary end-points were cure, overall response rates andsurvival. Toxicity was also evaluated in these mice, and a USFDA generalsafety study was conducted in healthy Balb/c mice. In Raji cell culture,the threshold and IC₅₀ concentrations for cytotoxic activity were 0.7and 2.5 nmol (pm/ml media), respectively. When compared to treatedJurkat's xenografts or untreated xenografts, Raji xenografts treatedwith the SHAL showed an 85% reduction in hazard of death (P=0.014; 95%confidence interval 32-95% reduction). There was no evidence fortoxicity even after i.p. doses 2000 times greater than the treatmentdose associated with cure of a majority of the mice with Rajixenografts. When compared with control groups, treatment selectivelyimproved response rates and survival in mice with HLA-DR10 expressinghuman lymphoma xenografts at doses not associated with adverse eventsand readily achievable in patients.

Introduction

Cell surface proteins, such as CD20, CD22 and MHCII HLA-DR, have provento be attractive targets for therapeutics for non-Hodgkin's lymphoma(NHL). One of these proteins, human leukocyte antigen (HLA) isupregulated on the surface of malignant B lymphocytes when compared tonormal B lymphocytes. Moreover, intracellular HLA-DR protein is evenmore abundant. HLA-DR proteins serve as transmembrane and cytoplasmicsignaling receptors and as peptide shuttles for the immune system(Leveille et al. (2002) Eur. J. Immunol. 32: 2282-2289; Klemm et al.(1998) Annu. Rev. Immunol., 16: 569-592; Lane et al. (1990) J. Immunol.,144: 3684-3692). The monoclonal antibody (MAb), Lym-1, binds to awell-characterized epitope on the β subunit of HLA-DR and reacts withlymphoma tissue from about 90 and 50% of patients with B-cell lymphomaand leukemia, respectively (DeNardo et al. (1998) J. Clin. Oncol., 16:3246-3256; Rose et al. 91996) Cancer Immunol. Immunother., 43: 26-30;Rose et al. (1999) Mol. Immunol., 36: 789-797). Although binding isrestricted to the cell surface (Epstein et al. (1987) Cancer Res 47:830-840), Lym-1 is highly active against malignant B cells in cultureand in mice (DeNardo et al. (2005) Clin. Cancer Res., 11: 7075-7079;Tobin et al. (2007) Leuk. Lymphoma 48: 944-956, 2007; Zhang et al.(2007) Cancer Biother. Radiopharm., 22: 342-356).

To mimic MAb binding to HLA-DR10, while decreasing size, a series ofselective high affinity ligands (SHALs)<5 kDa in size have beensynthesized to bind in the Lym-1 epitopic region of the β subunit ofHLA-DR protein based on in silico modeling and experimental studies.Bidentate versions of these novel nanomolecules showed many desiredcharacteristics in vitro and in mice (Balhorn et al. (2007) Clin. CancerRes., 13(Suppl. 18): S5621-S5628; DeNardo et al. (2007) J. Nucl. Med,48: 1338-1347) but had no antilymphoma activity (West et al. (2006)Cancer Biother. Radiopharm., 21: 645-654). Strikingly, a tridentate SHALcontaining the Ct ligand(3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionicacid) residualized inside and was cytotoxic for B-lymphoma cells inculture. Here, we titrate the cell selective antilymphoma activity ofthis SHAL, demonstrate its remarkable efficacy, selectivity and safetyin mice with human lymphoma xenografts and show electron microscopicevidence for SHAL-induced autophagy.

Materials and Methods

Reagents and Cell Lines.

Murine (Peregrine Pharmaceuticals, Tustin, Calif.) and chimeric (A.Epstein, Los Angeles, Calif.) Lym-1, that bind to the β subunit ofHLA-DR10 expressed on malignant B-cells (Epstein et al. (1987) CancerRes 47: 830-840), and HLA-DR10 protein isolated from Raji Burkitt'sB-cells using a Lym-1 affinity column, as described previously (Balhornet al. (2007) Clin. Cancer Res., 13(Suppl. 18): S5621-S5628) were usedas references. An HLA-DR10 expressing human B lymphoma cell line, Raji(American Type Culture Collection, Manassas, Va.), grown in RPMI-1640with 10% fetal calf serum, supplemented with sodium pyruvate,nonessential amino acids and antibiotics (Gibco/Invitrogen, Carlsbad,Calif.), at 37° C. in a humidified, 5% CO₂ atmosphere and anon-expressing human T lymphoma cell line, Jurkat's (American TypeCulture Collection, Manassas, Va.), grown as recommended by ATCC, wereused for these experiments.

Drug Design and Chemistry.

Using homology modeling, HLA-DR amino acid residues critical for Lym-1binding were mapped on a 3-D model of the HLA-DR10 β subunit (Balhorn etal. (2007) Clin. Cancer Res., 13(Suppl. 18): S5621-S5628). Cavitieswithin the Lym-1 epitopic surface of the protein were identified usingSPHGEN (Kuntz et al. (1982) J. Mol. Biol. 161: 269-288; Desjarlais etal. (1988) J. Med. Chem., 31: 722-729). After computational liganddocking for each of the cavities, a combination of NMR spectroscopy,surface plasmon resonance (BIAcore 3000; Biacore, Piscataway, N.J.) andcompetitive binding experiments were used to confirm ligand binding toHLA-DR10 protein. Sets of ligands were then linked to create a SHAL, asdescribed previously (Balhorn et al. (2007) Clin. Cancer Res., 13(Suppl.18): S5621-S5628). Each SHAL was synthesized on chlorotritylchlorideresin in a polyethylene column (Pierce Biotechnology, Inc.) using Fmocsolid phase chemistry to conjugate PEG monomer units and selectedligands through the alpha and epsilon amines of the N-terminal lysine.To synthesize the tridentate SHAL, a dabsylvaline (Dv) ligand wasattached to the terminal amine of a PEG spacer conjugated to the alphaamine of the 2nd lysine residue and a4-[4-(4-chlorobenzyl)piperazino]-3-nitrobenzenecarboxylic acid (Cb)ligand was attached to the terminal amine of a PEG conjugated to thealpha amine of the 3rd lysine. Lastly, the Ct ligand was also attachedto the epsilon amine of the 3rd lysine (FIG. 28). Conversion to DOTA (orbiotinylated) SHAL derivatives was accomplished, as describedpreviously. The reaction was monitored by analytical high performanceliquid chromatography (HPLC), and the DOTA (or biotinylated) derivativeswere purified using reverse phase, high performance liquidchromatography (RP-HPLC). Analytic electrospray ionization-massspectrometry (Agilent 1100 instrument, Waters Symmetry C18 column) wasused to confirm the elemental and mass composition of the SHAL(molecular weight within 0.07% of the theoretical molecular weight). Toexamine SHAL binding to isolated or recombinant HLA-DR10 protein,surface plasmon resonance experiments were conducted, as previouslydescribed (Id.). SHAL binding was better than nanomolar and was blockedby Lym-1 addition.

Cytotoxicity Assay.

Raji cells harvested in log growth phase were centrifuged, resuspendedin fresh media and counted in 10% trypan-blue dye to determine initialand subsequent viability, as previously described (West et al. (2006)Cancer Biother. Radiopharm., 21: 645-654). When untreated, cellscontinued to multiply and nonviable cells, initially <5% of total cells,remained so over the course of the assays. To titrate cytotoxicactivity, biotinylated tridentate SHAL at concentrations ranging between0 and 7 nM (pm/ml media) was incubated with 0.5×10₆ cells/ml at 37° C.in a 5% CO₂, humidified atmosphere (NAPCO, Portland, Oreg.). After 1, 2and 3 days, cells were resuspended in trypan blue dye and counted in ahemocytometer. The fractional viability and absolute number of viableand non-viable cells/ml were determined.

Efficacy and Toxicity in Mice.

Seven- to nine-week old female, athymic Balb/c nu/nu mice (HarlanSprague Dawley, Inc., Frederick, Md.) were maintained according toUniversity of California animal care guidelines under pathogen-freeconditions and on a normal ad libitum diet. Raji or Jurkat's cellsharvested in log phase and having >95% viability were implanted (6×10₆cells) subcutaneously in the lower abdomen 3-4 days after the mice wereirradiated (400 cGy) to suppress xenograft rejection. When xenograftsreached 20-500 mm₃ by caliper measurement, the mice were sorted byxenograft volume into 4 groups: SHAL-treated mice with Raji xenografts,SHAL-treated mice with Jurkat's xenografts, untreated mice with Rajixenografts or untreated mice with Jurkat's xenografts. Each mouse wasinjected with 100 μl of PBS or 100 ng DOTA-chelated tridentate SHAL i.p.on days 0, 7 and 14. Xenograft volume and survival were monitored for≧84 days. The mice were also weighed 2-3 times each week for 4 weeksthen weekly. Blood counts were measured prior to each SHAL or PBS doseand weekly for 4 weeks after the final dose using a Z Series CoulterCounter (Beckman Coulter Inc., Hialeah, Fla.) and a phase contrastmicroscope (Zeiss, Jena, Germany).

Xenograft volumes were calculated by the formula for hemiellipsoids(DeNardo et al. (1997) Clin. Cancer Res. 3: 71-79). Responses werecharacterized as a cure, xenograft completely disappeared and did notregrow by 84 days, the formal censor day (>210 days to the present)complete remission (CR), xenograft disappeared for at least 7 days, butregrew, and partial remission (PR), xenograft volume decreased by atleast 75% and remained stable for at least 7 days. All cures at 84 daysin SHAL-treated mice persisted over a 210-day observation time for themice. If a xenograft became >2000 mm₃, the mouse was euthanized inaccordance with University of California animal care guidelines andscored as a disease-related death.

Safety in Mice.

Each of 3 groups of 7 healthy Balb/c mice were given PBS, 20 or 200 μgof DOTA-chelated SHAL i.p., that is, 200 or 2,000 times the SHALefficacy dose. Immediately prior to intervention, the mice wereobserved, weighed and blood counts (red, white and platelets) obtained,then the mice were observed daily, weighed 3 times each week and bloodcounts measured weekly for 4 weeks after intervention.

Electron Microscopy (EM).

Raji xenografts (53-132 mm³) were harvested for EM from mice, untreated(control) and 2, 4 or 24 h after 100 ng of biotinylated SHAL. Xenograftswere fixed, as described (18), using 4% paraformaldehyde in 0.1 MSorenson's phosphate buffer, pH 7.35. After treating with 4% uranylacetate in 70% ethanol for one hour and LR White acrylic resinovernight, polymerization was achieved in a microwave (Pelco 34700BioWave, Pella Inc., Redding, Calif.). Ultra-thin sections (LeicaUltracut UCT, Leica, Vienna, Austria) were captured on gold grids,floated on Strepavidin-20 nm gold (BB International from Ted Pella Inc.)rinsed and stained with uranyl acetate and lead citrate before viewingusing a Philips CM120 Biotwin Lens (FEI, Hillsboro, Oreg., made inEindhoven, The Netherlands and Gatan MegaScan, model 794/20, digitalcamera (2K×2K), Pleasanton, Calif. Gatan BioScan, model 792, Pleasanton,Calif.).

Biostatistical Methods.

Initial analysis compared response rates by exact tests and survivaltimes by log-rank tests for untreated Raji and Jurkat's mice and foundno significant differences, so these groups were pooled for subsequentanalysis. Response rates for treated Raji, treated Jurkat's anduntreated mice were compared by χ² and exact tests. Survival times werecensored at 84 days for mice who had not died or been euthanized becauseof tumor growth before that time. Times were summarized descriptively byKaplan-Meier curves and median survival and 95% confidence limits wereobtained by the product-limit estimate. The two treated groups werecompared to the untreated using a log-rank test for homogeneity acrossgroups, followed by a proportional hazards model to estimate the effectof treatment compared to no treatment. Analyses were carried out usingSAS/SAT® software (SAS Institute, Inc, SAS/STAT Version 9.0. 2004. Cary,N.C.: SAS Institute). All tests were two-sided at level 0.05.

Results

Cytotoxicity Assay.

When untreated, non-viable (dead) Raji cells remained <5% of the totalcells over the 3 days of observation. Raji cells treated with SHAL (overa concentration range of 0-70 nM (pm/ml media)) showed more dead cells,both fractionally and absolutely, over the 3-day period when compared tothe untreated controls. The maximum number of non-viable cells wasobserved on day 2 at SHAL concentrations between 2.3 and 7 nM, and thethreshold and IC₅₀ SHAL cytotoxic concentrations were 0.7 and 2.5 nM(pm/ml media), respectively (FIG. 29).

Efficacy and Toxicity.

Regression in treated Raji xenografts typically began ˜7 and reachedcomplete remission ˜30 days after initial SHAL treatment (FIG. 30).Although censored at 84 days, all cures in SHAL-treated Raji xenograftshave persisted to the present, seemingly representing permanent cures.Because there were no significant differences in response and survivalof untreated Raji and Jurkat's mice (P>0.08) they were combined forcomparison to treated mice. Mice with Raji xenografts and treated withSHAL had a 69% cure rate and 77% overall response rate, significantlybetter than treated mice with Jurkat's xenografts (0%) and untreatedmice (15% response) (P<0.001) (Table 10). The median survival times were25 days for untreated mice and 21 days for treated mice with Jurkat'sxenografts. The median survival time for treated mice with Rajixenografts was not estimable because almost all mice were alive at 84days (FIG. 31). The proportional hazards model estimated that Raji micehad ˜85% reduction in hazard of death compared to untreated and treatedJurkat's mice (P<0.001).

TABLE 10 Athymic mice with lymphoma xenografts, SHAL treated oruntreated. Response (%) Treatment Group Mice Cure (%) ORR (%)^(a) Raji13 69 77 Jurkat's 9 0 0 Untreated 13 15 15 ^(a)Overall response rate.

Safety in Mice.

All of the mice in the general safety study, including those given 2000times the SHAL dose used in the efficacy trial, gained weight, showedstable blood counts and no adverse effects during 4 weeks ofobservation, thereby exceeding the requirements of the US FDA ‘generalsafety’ test guidelines.

Electron Microscopy.

Raji xenograft EM from untreated mice showed healthy cells havingwell-defined cellular and nuclear membranes, and cytoplasmic organelles,including reticular endothelium, Golgi, and liposomes (FIG. 32). Incontrast, Raji EM, harvested after SHAL treatment, were characterized byhighly condensed nuclear chromatin and fragmented organelles, findingsconsistent with autophagicy (programmed) cell death.

Discussion

Proteins on the surface of malignant cells have been used to identifythem and to serve as targets for therapy and imaging. The class II majorhistocompatibility, human leukocyte antigens (HLA) are abundant both onthe surface and inside malignant B-lymphocytes (Epstein et al. (1987)Cancer Res 47: 830-840). These proteins are major signaling receptorsfor cell death (Leveille et al. (2002) Eur. J. Immunol. 32: 2282-2289;Lane et al. (1990) J. Immunol. 144: 3684-3692). Whereas intact MAbsrecognize malignant cells selectively, size limits their blood clearanceand tissue penetration. Based on predictions from in silico modeling andfrom empiric testing, small organic ligands have been selected to bindto sites within the Lym-1 MAb epitopic region of HLA-DR10. By covalentlylinking sets of these ligands together, SHALs have been generated totarget B-cell derived lymphomas and leukemias (Hok et al. (2007)Bioconjug. Chem., 18: 912-921). These novel nanomolecules mimic theaffinity and selectivity of MAbs because of three-dimensionalinteractions made between multiple ligands in the SHAL and multiplesites located within the epitope of the target protein. SHALs are takenup rapidly by HLA-DR expressing xenografts and clear quickly from normaltissues (DeNardo et al. (2007) J. Nucl. Med, 48: 1338-1347) because theSHAL is ˜50 times smaller than an immunoglobulin G molecule.Histochemical analyses has shown that SHALs bind to NHL tissues frompatients and from mice (Balhorn et al. (2007) Clin. Cancer Res.,13(Suppl. 18): S5621-S5628). Previous studies have shown that the SHALsreadily enter cells, but they do not leave cells that express HLA-DR10and related HLA-DRs, behaving like a lobster and its trap.

These SHALs cross cell barriers efficiently, seemingly unaffected inHLA-DR10 expressing cells by cellular pumping mechanisms. SHALscontaining a Ct(3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionicacid) ligand residualize inside and exhibit potent antilymphoma activityagainst live human HLA-DR10 expressing lymphoma cells (DeNardo et al.(2007) J. Nucl. Med, 48: 1338-1347). The present experiments demonstratethat the Ct ligand containing tridentate SHAL that has an IC₅₀ of 2.5 nM(pm/ml media) for HLA-DR10 expressing cells in culture also exhibitsremarkable efficacy in mice with Raji xenografts. Permanent cures wereachieved in 69% of these mice at a SHAL treatment dose 2000-fold lessthan the maximum dose tested and confirmed to be safe in healthy mice.The antilymphoma effect was only observed in mice with xenograftsexpressing HLA-DR10; Jurkat's xenografts were not affected by SHALtreatment. Because mice treated with 2000-fold more SHAL did not exhibitsigns of adverse events, the SHAL seems to have an exceptional margin ofsafety as a potential therapeutic. It will be important in the future toconduct a dose response study with the tridentate SHAL in mice toascertain whether or not the cure rate can be increased with a largerSHAL dose.

Electron micrographs of xenograft tissue from mice treated with the SHALconfirmed the SHAL's cytotoxicity to the Raji lymphoma cells. Extensivevacuolization and loss of cell structure suggest the possibility thatSHAL binding to HLA-DR induced cell signaling that ultimately led toapoptosis and autophagic cell death. More extensive study will berequired to confirm this hypothesis and identify the mechanism of actionof the SHAL.

The results provide convincing evidence that SHALs have extraordinarypotential as novel nanomolecules for targeting lymphoma and leukemia formolecular therapy. SHALs represent an attractive alternative to theirbiological counterparts, because these chemicals are ˜50 times smaller,less expensive, easier to produce with consistency, stable and expectedto have a long shelf-life and be effective when given orally.Furthermore, SHAL-based therapeutics can transport and residualize otheragents near critical sites inside these malignant cells. The SHALproduction platform is efficient, flexible, and permits rapid synthesisand modifications.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method of inhibiting the growth orproliferation of a cancer cell that expresses an HLA-DR10 marker, saidmethod comprising: contacting said cancer with a selective high-affinitypolydentate ligand (SHAL), the SHAL comprising a first ligand, a secondligand and a third ligand: wherein said first ligand binds to a firstsite on a marker for said cancer cell linked to a second ligand thatbinds a second site on the same marker or a different marker for saidcancer cell wherein said first site and said second site are differentsites; and wherein at least said first ligand is a ligand selected fromthe group consisting of BOC-4-aminomethyl-L-Phe,4[[5-(Trifluoromethyl)pyridin-2-yl]oxy]phenyl]N-phenylcarbamate,(R)-2-[4-(5-chloro-3-fluoro-2-pyridyloxy)phenoxy]propionic acid,2-(((3-chloro-5(trifluoromethyl)pyridin-2-yloxy)phenoxy)methyl)acrylates,2-(((3-chloro-5(trifluoromethyl)pyridin-2-yloxy)phenyl)methyl)acrylates,2-(((3-chloro-5(trifluoromethyl)pyridin-2-yloxy)phenyl)methyl)acrylonitriles,3-(3-chloro-4-{[5-(trifluoromethyl)-2-pyridinyl]oxy}anilino)-3-oxopropanoicacid, Sethoxydim, Clethodim, 5-(Tetradecyloxy)-2-furoic acid,2-[(2,6-Dichlorophenyl)amino]benzeneacetic acid,2-[4-(4-Chlorophenoxy)phenoxy]propanoic acid,(RS)-2-{4-[3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy}propanoicacid, (RS)-2-[4-(6-chloro-1,3-benzoxazol-2-yloxy)phenoxy]propanoic acid,(RS)-2-[4-(2,4-dichlorophenoxy)phenoxy]propanoic acid,(RS)-2-{4-[5-(trifluoromethyl)-2-pyridyloxy]phenoxy}propanoic acid,(RS)-2-[4-(6-chloroquinoxalin-2-yloxy)phenoxy]propanoic acid,(RS)-2-[4-(α, α, α-trifluoro-p-tolyloxy)phenoxy]propanoic acid,5-([4,6-Dichlorotriazin-2-yl]amino)fluorescein hydrochloride,3-[N-(4-acetylphenyl)carbomoyl]pyridine-2-Carboxylic acid,3-(2-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy}anilino)-3-oxopropanoicacid, L-ornithine-beta-alanine,2-Methyl-1-(3-morpholinopropyl)-5-phenyl-1H-pyrrole-3-carboxylic acid,Hippuric acid, Hippuryl-D-lysine, and hippuryl-L-phenylalanine.
 2. Themethod of claim 1, wherein said first or second ligand is a smallorganic molecule.
 3. The method of claim 1, wherein said first ligandbinds a site that is different than the site bound by the second ligand.4. The method of claim 1, wherein said first site or second site is apocket on said marker.
 5. The method of claim 1, wherein said marker isan HLA-DR cell surface antigen.
 6. The method of claim 1, wherein saidfirst ligand and said second ligand bind sites within an epitoperecognized by the Lym-1 antibody.
 7. The method of claim 1, wherein saidsecond ligand is a ligand selected from Tables 1, 5, 6, 7, or
 8. 8. Themethod of claim 1, wherein said second ligand and said third ligand areindependently selected from the group of ligands found in from Tables 1,5, 6, 7, or
 8. 9. The method of claim 1, wherein: said first ligand is4-(Dimethylamino)azobenzene-4′-sulfonyl-L-valine (Dv); said secondligand is 4-[4-(4-chlorobenzyl)piperazino]-3-nitrobenzenecarboxylic acid(Cb); and said third ligand is selected from the group consisting of4[[5-(Trifluoromethyl)pyridin-2-yl]oxy]phenyl]N-phenylcarbamate,(R)-2-[4-(5-chloro-3-fluoro-2-pyridyloxy)phenoxy]propionic acid,2-(((3-chloro-5(trifluoromethyl)pyridin-2-yloxy)phenoxy)methyl)acrylates,2-(((3-chloro-5(trifluoromethyl)pyridin-2-yloxy)phenyl)methyl)acrylates,2-(((3-chloro-5(trifluoromethyl)pyridin-2-yloxy)phenyl)methyl)acrylonitriles,3-(3-chloro-4-{[5-(trifluoromethyl)-2-pyridinyl]oxy}anilino)-3-oxopropanoicacid, Sethoxydim, Clethodim, 5-(Tetradecyloxy)-2-furoic acid,2-[(2,6-Dichlorophenyl)amino]benzeneacetic acid,2-[4-(4-Chlorophenoxy)phenoxy]propanoic acid,(RS)-2-{4-[3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy}propanoicacid, (RS)-2-[4-(6-chloro-1,3-benzoxazol-2-yloxy)phenoxy]propanoic acid,(RS)-2-[4-(2,4-dichlorophenoxy)phenoxy]propanoic acid,(RS)-2-{4-[5-(trifluoromethyl)-2-pyridyloxy]phenoxy}propanoic acid,(RS)-2-[4-(6-chloroquinoxalin-2-yloxy)phenoxy]propanoic acid, and(RS)-2-[4-(α, α, α-trifluoro-p-tolyloxy)phenoxy]propanoic acid.
 10. Themethod of claim 1, wherein said SHAL is a tridentate SHAL described inTable
 2. 11. The method of claim 1, wherein said first ligand is joinedto said second ligand by a linker selected from the group consisting ofa PEG type linker, a peptide or peptide analog linker, an avidin/biotinlinker, a straight chain carbon linker, a heterocyclic linker, abranched carbon linker, a dendrimer, a nucleic acid linker, a sugar orcarbohydrate linker, a thiol linker, an ester linker, a linkercomprising an amine, and a linker comprising a carboxyl.
 12. The methodof claim 1, wherein said first ligand, said second ligand and said thirdligand are joined to each other by a linker comprising a moiety selectedfrom the group consisting of a PEG type linker, a peptide linker, anavidin/biotin linker, a straight chain carbon linker, a heterocycliclinker, a branched carbon linker, a dendrimer, a nucleic acid linker, asugar or carbohydrate linker, a thiol linker, an ester linker, a linkercomprising an amine, and a linker comprising a carboxyl.
 13. The methodof claim 11 or 12, wherein said linker comprises polyethyleneglycol. 14.The method of claim 11 or 12, wherein said linker comprisespolyethyleneglycol and a lysine.
 15. The method of claim 1, wherein saidfirst ligand is 4-(Dimethylamino)azobenzene-4′-sulfonyl-L-valine (Dv);said second ligand is4-[4-(4-chlorobenzyl)piperazino]-3-nitrobenzenecarboxylic acid (Cb); andsaid third ligand is selected from the group consisting of3-(2-([3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanoicacid (Ct), or an analogue thereof.
 16. The method of claim 1 or 15,wherein said first ligand, said second ligand and said third ligand arejoined to each other by a linker comprising a moiety selected from thegroup consisting of a PEG type linker, a peptide linker, anavidin/biotin linker, a straight chain carbon linker, a heterocycliclinker, a branched carbon linker, a dendrimer, a nucleic acid linker, asugar or carbohydrate linker, a thiol linker, an ester linker, a linkercomprising an amine, and a linker comprising a carboxyl.
 17. The methodof claim 16, wherein said linker comprises polyethyleneglycol.
 18. Themethod of claim 16, wherein said linker comprises polyethyleneglycol anda lysine.
 19. The method of claim 1, wherein said SHAL comprises thestructure:

wherein R¹ is selected from the group consisting of COOH, a linker, aneffector, and a linker attached to an effector.
 20. The method of claim1, wherein said SHAL comprises the structure:

wherein R² comprises an effector.